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Identification of four novel HLA-DOA alleles, DOA*010106, DOA*0102, DOA*0103, and DOA*0104N, by sequence-based typing*.

DOA sequences are currently known to have identical protein sequences. However, in this study, we report four novel allele types of human leucocyte antigen-DOA, including one synonymous and three non- synonymous amino acid changes from the Korean population. DOA*010106 has identical protein sequence with previously known DOA*010103 except one nucleotide difference at codon 45 (TCG-->TCA). In contrast, DOA*0102 and DOA*0103 have a sequence change at codon 99 (CTG-->GTG) and codon 105 (CGC-->TGC), causing non-synonymous amino acid changes, Leu99Val and Arg105Cys, respectively. In addition, DOA*0104N has a sequence deletion at codon 36 (CCC-->CC-), resulting in a frame shift leading to a stop codon at codon 62.

Alleles↗

Genetically diverse group A streptococci from children in far-western Nepal share high genetic relatedness with isolates from other countries.

The genetic diversity of group A streptococci (GAS) throughout much of the world has not been adequately explored. To assess genetic variation among GAS in western Nepal, 120 noninvasive GAS, collected from eight different villages, were genetically characterized using emm typing, sof sequencing, and multilocus sequence typing (MLST). A high level of genetic diversity was observed among these isolates, with 51 genotypes based upon 51 multilocus sequence types (STs), 45 emm sequence types, and 28 sof sequence types. On the basis of shared ST-emm and sof-emm associations, 40 of the 51 genotypes were identical or highly related to genotypes characterized from locations outside of Nepal, even though most of the emm sequence and clonal types are rare among GAS within the United States. When analyzing all known STs highly related to Nepal STs, only one example of similar STs shared between a sof PCR-positive strain and a sof PCR-negative strain was observed. Since previous data indicate free exchange of MLST loci between sof-positive and sof-negative strains, there is possibly selection against the expansion of subclones resulting from horizontal transfers of sof or emm genes between sof-positive and sof-negative strains. All 45 emm types encountered in Nepal have also been documented from other countries. These data, together with data encompassing the past decade of emm type surveillance, support the possibility that most existing GAS emm types have been discovered. Similarly, since most (40/51) strain types were highly related to strains found elsewhere, it is likely that a major fraction of the existing GAS clonal complexes have been discovered.

Antigens, Bacterial↗

Completion of the DNA sequence of mouse adenovirus type 1: sequence of E2B, L1, and L2 (18-51 map units).

The DNA sequence of 9991 nt, corresponding to 18-51 map units of mouse adenovirus type 1 (MAV-1), was determined, completing the sequence of the Larsen strain of MAV-1. The length of the complete MAV-1 genome is 30,946 nucleotides, consistent with previous experimental estimates. The 18-51 map unit region encodes early region 2B proteins necessary for adenoviral replication as well as late region L1 and L2 structural and packaging proteins. Sequence comparison in this region with human adenoviruses indicates broad similarities, including colinear preservation of all recognized open reading frames (ORFs), with highest amino acid identity occurring in the DNA polymerase and polypeptide III (penton base subunit) ORFs. Virus-associated (VA) RNA is not encoded in the region where VA RNAs are found in the human adenoviruses, between E2B and L1, nor is it encoded anywhere in the entire MAV-1 genome. The MAV-1 polypeptide III lacks the arginine-glycine-aspartic acid (RGD) motif which is involved in an association with cell-surface integrins. Only one RGD sequence is found in an identified coding region in the entire MAV-1 genome. Similar to the porcine adenovirus, this RGD sequence is found in the C-terminus of the MAV-1 fiber protein.

Adenovirus E2 Proteins↗

Sequence-based typing of the complete coding sequence of DQA1 and phenotype frequencies in the Dutch Caucasian population.

Typing of DQA1 by sequencing has been a challenge because of a 3-nucleotide deletion in exon 2 in half of the alleles. Furthermore, 19 of the 28 alleles cannot be identified on basis of exon 2 alone, but need additional exon information. With the sequencing strategy presented here the complete exons 1-4 are sequenced heterozygously, enabling identification of all DQA1 alleles by sequence-based typing (SBT). Exons 1-4 were amplified and sequenced separately, the combined sequences were used for automated allele assignment. The method was validated by typing 21 individuals with all possible different allele group combinations. In addition 26 quality control samples were correctly typed by this method. To determine the phenotype frequencies 155 unrelated Dutch Caucasian individuals were DQA1 typed. In total 15 known and two new DQA1 alleles were identified. DQA1*0103 and *0505 were the most frequent alleles with phenotype frequencies of 30% and 29%, respectively. The SBT method presented here is an improvement compared to already existing protocols in that the complete exon sequence is obtained for all coding exons, using identical polymerase chain reaction conditions. Furthermore, all exons are sequenced heterozygously, facilitating allele assignment and reducing the number of amplification reactions.

Alleles↗

Two international methicillin-resistant Staphylococcus aureus clones endemic in a university hospital in Patras, Greece.

Pulsed-field gel electrophoresis (PFGE) of SmaI macrofragments and hybridization of ClaI digests with the mecA- and Tn554-specific DNA probes were used to define the endemic clones of methicillin-resistant Staphylococcus aureus (MRSA) among strains collected in 1993 and 1998 to 2000 at the University Hospital of Patras, Patras, Greece. Representatives of each clonal type were analyzed by spaA typing, multilocus sequence typing (MLST), and staphylococcal chromosomal cassette mec (SCCmec) typing. The results indicated the existence of two successive international MRSA clones: (i) a clonal type with PFGE type A, sequence type (ST) 30 (ST30), and SCCmec type IV, which was very similar to a clone widely spread in the United Kingdom, Mexico, and Finland, and (ii) a clonal type with PFGE type B, ST239, and SCCmec III, which was related to the Brazilian clone. Both clones seem to be widespread in Greece as well. A novel MRSA clone is also described and is characterized by a new MLST type (ST80) associated with SCCmec type IV and with the presence of Panton-Valentine leukocidin genes.

Bacterial Typing Techniques↗

Molecular characterisation of a dominant methicillin-resistant Staphylococcus aureus (MRSA) clone in a Mexican hospital (1999-2003).

Methicillin-resistant Staphylococcus aureus (MRSA) isolates (n = 216), collected between January 1999 and May 2003 in a tertiary-care university hospital in Guadalajara, Mexico, were characterised by antibiotype, pulsed-field gel electrophoresis (PFGE) of SmaI macrorestriction fragments, and hybridisation of ClaI digests with mecA- and Tn554-specific DNA probes. Representatives of the single clonal type found were analysed by spa typing, multilocus sequence typing and staphylococcal chromosomal cassette mec (SCCmec) typing, and were tested for the presence of 22 virulence determinants and agr type. A single PFGE pattern was identified, with minor variations over time, with spa type 2, sequence type 5, SCCmec type II, agr type 2 and the presence of the enterotoxin genes seg and sei, the gamma-haemolysin variant gene hlg-v and the leukocidin lukE-lukD genes. In addition, the isolates showed antimicrobial resistance to beta-lactams, macrolides, chloramphenicol and imipenem, and susceptibility to gentamicin, rifampicin, trimethoprim-sulphamethoxazole and vancomycin. Following its appearance in 1997, this clone spread within the hospital, and is now present in most of the hospital units and wards.

Adolescent↗

Universal versus targeted chlorhexidine and mupirocin decolonisation and clinical and molecular epidemiology of Staphylococcus epidermidis bloodstream infections in patients in intensive care in Scotland, UK: a controlled time-series and longitudinal genotypic study.

BACKGROUND: There are concerns that biocide skin and mucous membrane decolonisation, which is widely used to prevent health-care-associated infections in intensive care units (ICUs), might select for multidrug-resistant pathogens. We aimed to evaluate the effects of de-escalating from universal to targeted skin and nasal decolonisation on Staphylococcus epidermidis bloodstream infections (SE-BSI). METHODS: We did a retrospective, before-after-control-impact time-series analysis and longitudinal genotypic study in two ICUs with divergent decolonisation practice in tertiary care hospitals of adjacent health boards in Scotland, UK. Participants were aged at least 16 years and admitted between July 1, 2009, and Feb 28, 2022. There were no exclusion criteria for the study. In ICU one (intervention site) universal decolonisation in all admissions was de-escalated to targeted decolonisation of meticillin-resistant Staphylococcus aureus (MRSA) carriers on Feb 1, 2019, while in ICU two (control site) targeted decolonisation was applied throughout. We collected bloodstream infection data from all causes, including clinically significant SE-BSI. Antimicrobial susceptibility testing was used to define meticillin-resistant S epidermidis (MRSE) and chlorhexidine susceptibility. We used multilocus sequence typing to identify sequence types from archived SE-BSI isolates. Whole-genome sequencing was applied to a sample from ICU one. The primary outcomes were incidence densities of all bloodstream infections, SE-BSI, and meticillin-resistant S epidermidis bloodstream infections (MRSE-BSI), and the percentage probability that SE-BSI were MRSE-BSI. The effects of de-escalation on primary outcomes were estimated by differences between the intervention and control sites, before and after de-escalation, using a before-after-control-impact time-series design. Secondary outcomes included the proportion of multidrug resistant sequence types, carriage of mobile genetic elements and genes for multidrug resistance and biofilm production. FINDINGS: Between July 1, 2009, and Feb 28, 2022, S epidermidis was identified in 334 (45%) of 735 bloodstream infections in ICU one, of which 197 occurred before the de-escalation intervention in Feb 1, 2019, and S epidermidis was identified in 167 (60%) of 278 bloodstream infections in ICU two. There was no increase in all bloodstream infection incidence coinciding with de-escalation in ICU one, whereas MRSE-BSI incidence declined significantly from 10·4 cases per 1000 occupied bed days (OBDs; 95% credible interval [CrI] 7·2-15·4) to 4·3 cases per 1000 OBDs (2·5-6·7), as did the percentage probability of MRSE (from 89·2%, 95% CrI 77·8-96·5 to 56·7%, 34·3-77·5%). No significant changes in the primary outcomes were seen in ICU two. MRSE-BSI incidence density was positively associated with chlorhexidine use, but not mupirocin use. De-escalation was associated with a reduced proportion of SE-BSI due to multidrug-resistant sequence types and reduced carriage of mobile genetic elements and genes for multidrug resistance and biofilm production, as observed by multi-locus sequence typing and whole genome sequencing. INTERPRETATION: In ICU settings with low MRSA incidence, the benefits of universal decolonisation should be balanced against the risks of selecting MRSE sequence types adapted for invasive and device-associated infection. FUNDING: National Health Service Grampian Charity.

Humans↗

Selection of unrelated bone marrow donors by PCR-SSP typing and subsequent nonradioactive sequence-based typing for HLA DRB1/3/4/5, DQB1, and DPB1 alleles.

HLA incompatibility between bone marrow recipients and unrelated donors is one of the main obstacles in bone marrow transplantation. HLA class I and generic class II DR and DQ typing is generally performed by serology. Precise subtyping of HLA class II genes, however, can only be achieved by molecular genetic methods. Here, the final selection of serologically pretyped unrelated bone marrow donors by confirmatory PCR-SSP (PCR-sequence-specific primers) typing and subsequent nucleic acid sequence analysis of the second exon of DRB1, DRB3, DRB4, DRB5, DQB1, and DPB1 alleles is presented. Serologically identical potential marrow donors and their corresponding recipients were analyzed for HLA-DRB identity by PCR-SSP analysis. After solid-phase single-strand separation, direct sequencing of the allele- or group-specific DRB amplified products was performed by applying fluorophorlabelled sequencing primers. Electrophoretically separated sequencing products were detected by means of an automated DNA sequencer. Group-specific amplification and sequencing of DQB1 alleles was carried out for all potential bone marrow donors and recipients, while only the final donor-recipient pair was analyzed for DPB1 alleles. Thus, the presented amplification strategy in combination with direct sequencing of PCR products allows matching of bone marrow transplant pairs with the highest degree of reliability for the assessment of HLA class II identity.

Base Sequence↗

Evidence of paternal transmission of mitochondrial DNA in a nucleus-cytoplasm hybrid of timopheevi wheat.

Structural heterogeneity depicted as heteroplasmy of the mitochondrial (mt) transcriptional unit of nad3-orf156 (atp8) was studied in a nucleus-cytoplasm (NC) hybrid of Triticum timopheevi with the D plasmon from the maternal Aegilops squarrosa and compared with that of the parental lines. The tetraploid NC hybrid and the parental lines both showed varying degrees of heteroplasmy in this mtDNA region. The G plasmon of the paternal T. timopheevi possessed five sequence types, while two sequence types were detected in the D plasmon of Ae. squarrosa. The NC hybrid possessed all the five sequence types identical to those of the paternal parent in a 30% relative stochiometry. The remaining 70% comprised only one of the two maternal sequence types, suggestive of strong and selective NC interaction. No novel sequence types were detected and the relative stoichiometries of the paternal sequence types were conserved in the NC hybrid. No paternal-identical or -related sequences were detected in the maternal D plasmon. These results provide evidence of the paternal transmission of the mtDNA and possibly account for the origin of the observed mtDNA heteroplasmy in the NC hybrid.

Base Sequence↗

High-resolution HLA typing for the DQB1 gene by sequence-based typing.

The ideal high-resolution typing strategy for polymorphic genes is sequence-based typing. SBT of genomic DNA has been developed for the HLA class II genes DRB1, DRB3/4/5 and DPB1. For the DQB1 gene the sequence-based typing method was shown to cause a number of problems. To resolve those problems, different primers to amplify and sequence exon 2 of DQB1 were designed and tested. With several primer combinations, preferential amplification was observed in individuals heterozygous for DQB1*02/*03 and DQB1*02/*04. The preference was for DQB1*02 in many instances but could also be demonstrated for DQB1*03 or *04 and resulted occasionally in allelic drop-out. The best primer combination was selected and successfully used to type individuals heterozygous for DQB1*02, *03 and *04. To distinguish DQB1*0201 and *0202, primers for amplification and sequencing of exon 3 were developed and correct subtyping was obtained. The ambiguous typing DQB1*0301/*0302 and DQB1*0303/*0304 was resolved by allele-specific amplification and sequencing. A total of 258 individuals were fully typed for their DQB1 subtypes. All samples had been previously typed by PCR-SSP and serology. Concordant typing results were obtained for all individuals tested. The DQB1 alleles detected included *0501, *0502, *0503, *0601, *0602, *0603, *0604, *0609, *0201, *0202, *0301, *0302, *0303, *0304, *0401 and *0402. Sequence-based typing of the DQB1 gene proved a reliable typing strategy for assignment of the different DQB1 alleles after intensive selection of primers and test conditions.

DNA Primers↗

Limited allelic diversity of Plasmodium falciparum merozoite surface protein 1 gene from populations in the Solomon Islands.

Meiotic recombination generates allelic diversity in the Plasmodium falciparum merozoite surface protein 1 (msp1) gene. In this study, we monitored recombination-based diversity of msp1 in Guadalcanal, the Solomon Islands, where malaria transmission is high. We identified 5' recombinant types, 3' sequence types, and msp1 haplotypes (unique associations of 5' recombinant types and 3' sequence types), and compared them with those from areas of low transmission in Thailand and Vanuatu. The mean number of 5' recombinant types per person (multiplicity) was lower in Guadalcanal than in Thailand. Guadalcanal populations had 6-8 msp1 haplotypes; the numbers are comparable to Vanuatu but much lower than in Thailand. There were marked geographic differences in distribution of msp1 haplo-types. Linkage disequilibrium in msp1 was stronger in Guadalcanal than in Thailand, suggesting limited recombination events in the Solomon Islands. We suggest that the frequency of recombination events in msp1 is determined not only by transmission intensity but by the number of msp1 alleles prevalent and multiplicity of infections.

Adolescent↗

Identification of multiple genital HPV types and sequence variants by consensus and nested type-specific PCR coupled with cycle sequencing.

Consensus and type-specific HPV primers were employed for PCR and cycle sequencing of genital HPVs in scrapings and colposcopically directed biopsies of the cervix from a cohort of 188 female sex workers. A total of 27 individuals tested positive for a broad spectrum of HPV types, including HPVs 6b, 16, 18, 31, 33, 34, 35, 45, 56 and 58, as well as a new HPV type, with seven individuals displaying dual infections. Good correlation between the results of individually paired samples was observed. A HPV 16 primer biotinylated at the 5' end was also used as a probe, which could successfully detect amplified products of HPV 16 but not other HPV types tested by an automated ELISA detection system. DNA sequence analysis revealed several HPV sequence variants that harbored mutations, especially in the E6 gene, many of which culminated in non-conservative amino acid substitutions in the transforming E6 oncoprotein. Such an approach of coupling PCR with cycle sequencing permits the determination of many known and even novel HPV types associated with varying degrees of risk to cervical carcinogenesis, and enables the identification of HPV sequence variants of putative biological and clinical significance, thus justifying its utility as an adjunct tool to complement cervical cytology and colposcopy. This study also emphasises the need for educational, interventional and behavioral modification to minimise HPV transmission, such as through consistent condom usage among sex workers.

Cervix Uteri↗

New Delhi metallo-β-lactamase-producing Acinetobacter baumannii in the USA from October, 2013, to March, 2022: a retrospective molecular epidemiological analysis.

BACKGROUND: Most US carbapenem-resistant Acinetobacter baumannii (CRAB) isolates harbour carbapenem-hydrolysing class D β-lactamases. Other carbapenemases, such as New Delhi metallo-β-lactamase (NDM), are uncommon but emerging. We describe the epidemiology of NDM-producing CRAB reported to the US Centers for Disease Control and Prevention (CDC). METHODS: We defined cases as A baumannii with blaNDM confirmed by molecular testing and isolated from any specimen source from a patient in the USA between Oct 1, 2013, and March 31, 2022, and passively reported to the CDC from regional, state, local public health, and CDC laboratories. Epidemiologically linked cases had epidemiological linkage (eg, overlapping health-care facility stay) with one or more other cases. We assessed case relatedness through analysis of whole-genome sequence data using traditional multilocus sequence typing (MLST; Oxford scheme [sequence typeOX]) and core genome MLST. To understand the potential origins of NDM-CRAB in the USA, we compared sequences of cases to US CRAB without NDM and to NDM-CRAB from non-US locations. FINDINGS: We identified 327 NDM-CRAB cases from 264 patients in 21 US states. Among patients with available epidemiological information, 192 (90%) of 214 had epidemiological linkage to at least one additional case and 13 (7%) of 193 were hospitalised outside the USA 12 months or less before index specimen collection. Five regionally distinct sequence type clusters were identified among the 264 case patients; three (sequence type OX218, sequence type OX281, and sequence type OX1697) were closely related to international NDM-CRAB isolates. INTERPRETATION: We identified regionally distinct NDM-CRAB strains, suggesting localised transmission in the USA. Some NDM-CRAB strains in the USA are closely related to strains identified outside the USA, suggesting that spread followed importation. FUNDING: None.

Acinetobacter baumannii↗

HLA-DQB1 sequencing-based typing using newly identified conserved nucleotide sequences in introns 1 and 2.

Sequencing-based typing (SBT) human leukocyte antigen (HLA) class I and II genes should examine entire exon sequences where polymorphisms lie. Primers for the amplification of complete exons therefore anneal in introns and their design relies on accurate intron sequences being available. We decided to develop a SBT method for HLA-DQB1 using amplification primers which anneal in introns 1 and 2, yet the amount of intron sequence data previously available in databases was sparse. Therefore, we undertook a systematic sequencing of introns 1 and 2 using DNA from cell lines homozygous for DQB1. This study confirmed an earlier report that the non-coding regions of this gene are the most polymorphic seen in the human genome. Intron sequences within an allele group were largely identical, the exceptions being DQB1*0301 differing from other DQB1*03 allele groups and DQB1*0601 differing from all other DQB1*06 alleles. A retroviral Alu element, related to the AluYa5a2 subfamily, was identified uniquely inserted in intron 2 of DQB1*02 alleles. For the typing approach, six amplification primers were designed based on conserved allele group sequences covering all of the HLA DQB antigens, and two sequencing primers were also designed which anneal in intron 2. This method has proved to be very robust and has been used as part of a referral DNA sequencing service for a number of years.

Alleles↗

Different clonal complexes of methicillin-resistant Staphylococcus aureus are disseminated in the Euregio Meuse-Rhine region.

The Euregio Meuse-Rhine (EMR) is formed by the border regions of Belgium, Germany, and The Netherlands. Cross-border health care requires infection control measures, in particular since the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) differs among the three countries. To investigate the dissemination of MRSA in the EMR, 152 MRSA isolates were characterized by pulsed-field gel electrophoresis (PFGE), SCCmec typing, and multilocus sequence typing. PFGE revealed major clonal groups A, G, L, and Q, suggesting dissemination of MRSA in the EMR. Group A harbored mainly SCCmec type III and sequence types (STs) 239 and 241. The majority of the strains from group G harbored SCCmec type I and ST8 and ST247, whereas most strains from group L carried either SCCmec type IV or type I. Within group L, ST8 and ST228 were found, belonging to clonal complexes 8 and 5, respectively. Most strains from group Q included SCCmec type II and were sequence typed as ST225. Both ST225-MRSA-II and ST241-MRSA-III were novel findings in Germany. In addition, the SCCmec type of two isolates has not been described previously. One strain was classified as SCCmec type III but harbored the pls gene and the dcs region. Another strain was characterized as SCCmec type IV but lacked the dcs region. In addition, one isolate harbored both SCCmec type V and Panton-Valentine leukocidin. Finally, the SCCmec type of the strains was found to be correlated with the antibiotic susceptibility pattern.

Belgium↗

High-resolution HLA typing for the DRB3/4/5 genes by sequence-based typing.

The high degree of polymorphism of the HLA genes at the nucleotide sequence level has proven sequence-based typing a major typing strategy. For DRB1 the allelic variability is predominantly present in the second exon and by DNA sequencing of exon 2 all hitherto known DRB1 alleles can be detected. For the associated genes DRB3, DRB4 and DRB5 the situation is slightly different. Allelic differences are not limited to exon 2 and the sequence of exon 3 and sometimes exon 4 is needed for complete subtyping. Oligonucleotides to amplify the exons needed for subtyping of DRB3, DRB4 and DRB5 were designed. Gene-specific products were generated to make simultaneous detection of alleles in heterozygous combinations possible. In this way 238 individuals were fully typed for their DRB3, 4 and 5 subtypes. Additional samples were typed for only one of the genes. All samples had been previously typed by PCR-SSP. Concordant typing results were obtained for all individuals tested. The DRB3 alleles typed for included *0101, *0201, *0202 and *0301, for DRB4 they were *01011, *0102 and *0103 and for DRB5 *0101, *0102, *0103, *0105, *0201, *0202 and *0203. All alleles were easily detected by the protocol described except for DRB5*0201. Sequencing of exon 3 and 4 of the DRB5*0201 allele showed this allele to be a sequencing error and the sequences obtained were identical to the exon 2, 3 and 4 sequences of DRB5*0202. Two new alleles were identified in the samples studied, DRB4*0105 and DRB3*0207. Sequence based typing has been recognized as a valuable tool for HLA typing of DRB1, DQB1 and DPB1 since several years. It is shown to be a superior typing method as well in the detection of the different DRB3, 4 and 5 subtypes.

Alleles↗

Frequent recovery of a single clonal type of multidrug-resistant Staphylococcus aureus from patients in two hospitals in Taiwan and China.

One hundred thirty-two methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered from patients with S. aureus infections between January 1998 and February 1999 in two hospitals, one located in Taipei, Taiwan, and another in Nanjing, People's Republic of China, were examined for antibiotic susceptibility and for clonal type by a combination of three methods: hybridization of ClaI restriction digests with mecA- and Tn554-specific DNA probes and pulsed-field gel electrophoresis of chromosomal SmaI digests. Selected isolates representing each clonal type were also analyzed by spaA typing, multilocus sequence typing, and a multiplex PCR method capable of identifying the structural type of the staphylococcal cassette chromosome mec (SCCmec) carried by the bacteria. The overwhelming majority of isolates (126 of 132 or 95%) belonged to minor variants of a single clonal type resembling the Brazilian and Hungarian epidemic MRSA clones, which showed a common spaA type and which were either sequence type 239 (ST239) or ST241 (a single-locus variant of ST239) in association with SCCmec type III or IIIA.

Anti-Bacterial Agents↗

Sequence-based typing of Mycoplasma genitalium reveals sexual transmission.

Mycoplasma genitalium causes male nonchlamydial, nongonococcal urethritis and is associated with cervicitis and pelvic inflammatory disease in women. Epidemiological studies indicate that M. genitalium is sexually transmitted, and the aim of the present study was to further substantiate this by means of a DNA typing system. A typing assay based on a diagnostic mgpB gene PCR was developed, evaluated, and applied directly to urogenital specimens. The assay had a low limit of detection and hence a high typeability. Sequences of isolates from 52 unrelated patients were divided into 29 different sequence types, giving a discriminatory index of 0.95. Two to six M. genitalium-positive specimens were collected from each of 44 patients over a median interval of 56 days (range, 11 to 1,395). Forty had the same sequence type in consecutive specimens. Specimens collected from two men were repeatedly positive at intervals of 472 and 1,395 days, respectively, but the sequence types had changed. A new strain was introduced in one sexual dyad, and the sequence types changed subsequently. Seventy-nine M. genitalium-positive specimens from 19 couples were investigated, and all partners initially had concordant sequence types, but one couple had discordant types at one time point before a newly introduced strain took over. The present typing system is simple and reproducible and has an excellent discriminatory capacity which might prove useful in studies of sexual networks and for evaluation of treatment failures. In the laboratory, this system may document the uniqueness of newly isolated M. genitalium strains.

Bacterial Typing Techniques↗