typing of isolates and positive clinical samples is necessary to support epidemiologic data for the detection of outbreaks and understand the transmission of infection . in contrast to typing based on sequence differences in the p1 gene of m.  pneumoniae , multiple - locus variable - number tandem repeat analysis ( mlva ) is reportedly highly discriminatory   and is now increasingly in use for strain characterization internationally [ 411 ] . investigating the five loci selected ( mpn1 , mpn1316 ) , it has been reported that the mpn1 locus is not stable , thus calling into question the reliability of the marker . therefore , several authors have proposed an alteration to the naming system to reflect this . despite the availability of general guidelines for the mlva procedure , specific internationally agreed guidelines for the execution and interpretation of mlva of m.  pneumoniae are not yet available . in this study , 24 m.  pneumoniae clinical isolates were included , as well as the reference strain m129 ( atcc 29342 ) . the clinical isolates , all derived from sputum specimens , were obtained from the public health england respiratory and vaccine preventable bacteria reference unit culture collection . dna was extracted from bacterial cultures in mycoplasma liquid medium ( mycoplasma experience ltd . , uk ) using the magna pure compact nucleic acid isolation kit i ( roche ) . the commercial quantitative type strain nctc10119 fh ( minerva biolabs ) was used to determine sensitivity of mlva at four dilutions ( 1000 , 100 , 10 and 1  copy/l ) . mlva was performed in a blinded manner using a previously described method   in six international laboratories ( china , england , france , germany , netherlands , united states of america ) . results were collated , including fragment size , calculated mlva repeat number and mlva profile . no guidelines were given to the participating laboratories other than that already available in the literature . naming of profiles was based on the method in which naming is based on a string of allele numbers in the order mpn1 , mpn13 , mpn14 , mpn15 , mpn16 showing the actual number of repeats at each locus . regarding fragment sizes , excellent parity was seen among laboratories , with < 7  bp difference in fragment sizes between all dna samples and all laboratories . table  1 includes expected fragment size and repeat numbers in order to clarify predicted fragment size and repeat number . regarding the fragment repeat numbers , a total of three errors on assigning and collating the mlva repeat number from the accurate fragment size ( transcription errors ) were noted from two separate laboratories ( table  2 ) . in addition , there was an inconsistency in the results reported for two of the loci , mpn13 and mpn15 . this was due to a different interpretation of fragment repeat number when encountering a point number . specifically , four of the laboratories rounded 3.2 copies up to 4 repeats for mpn13 , whereas two other laboratories rounded < 3.5 down to 3 repeats . this highlights the need for an internationally agreed protocol regarding the interpretation of mlva repeat numbers . in addition , one laboratory made calculating errors linked to the determination of the sequence of mpn15 . the mpn15 sequence was manually determined as tgtccatttttacttccatcat , in contrast to the accurate ttgtccattttttcttccatc sequence calculated using tandem repeat finder software with settings match , mismatch , indel of ( 2,3,5 ) . it should be noted that the use of settings other than ( 2,3,5 ) can give alternative repeat sequence and length for  some loci . for example , using settings ( 2,7,7 ) for m.  pneumoniae m129 , the mpn15 repeat would be only 20  bp with a different sequence ( ttgtccattttttttccatc instead of ttgtccattttttcttccatc ) . excluding the three transcription errors , and after correcting for interpretation differences by rounding up partial repeat numbers to the next integer value , all laboratories determined identical fragment repeat numbers for the m129 strain , and 20 of the 24 clinical isolates gave consistent fragment repeat numbers ( table  2 ) . actual technical differences were seen in only four samples : samples 8 , 10 , 21 and 30 . interestingly , these samples had lower - than - average dna concentration on initial dna extraction ( less than 3  g / ml dna , compared to 7  g / ml for the other samples ) . to compare the sensitivity , a serial dilution of the nctc10119 however , only three laboratories obtained a full profile , while the other three reported partial profiles for the 100  copies/l standard ; two of these partial profiles differed in the repeat number for mpn14 . a full profile was obtained with the 10  copies/l standard by only two laboratories . none of the laboratories obtained a full profile for the lowest dilution tested ( 1  copy/l ) . when examining both the serial dilution and the low - loaded sample results , it was apparent that in laboratory 5 , which increased the number of amplification cycles to 45 for samples that gave poor results with 25 cycles of amplification , the typing method showed a greater sensitivity . it indicates that comparison of existing published mlva data between laboratories may be flawed in some cases , diminishing reliability of strain investigation involving more than one laboratory . the following recommendations are considered pertinent by our collaborative group in enabling standardization of interpretive data . first , predicted fragment sizes and repeat numbers should be assigned using the information provided in table  1 . sequence of repeat fragments listed in table  1 should be considered as the sequence of interest . second , if tandem repeat finder software is used ( http://tandem.bu.edu/trf/trf.html )   to determine repeat numbers , the following settings should be used : match , mismatch , indel ( 2,3,5 ) . third , the repeat number should be expressed as whole integers , and partial sequences should be rounded up to the next integer number . however , as previously reported , rounding the partial number of repeats up and not down will avoid rounding down to zero a repeat number such as 0.7 , which is ambiguous , as it may be understood as lack of repeat. thus , using a  rounding up convention , zero will unambiguously be defined as  lack of repeat. moreover , by retaining a rounding - up approach , future data will correspond with historical data in previous publications related to m.  pneumoniae mlva typing . fourth , the mpn1 target should be removed from future analyses due to its instability . the identification of additional mlva targets that have greater stability than target mpn1 and enable greater discrimination power than mpn16 should be advanced . with the removal of the mpn1 allele , adoption of the following naming system is recommended : mlva-1 , -2 , -3 and -4 , where each digit corresponds to repeat numbers at loci mpn13 , mpn14 , mpn15 and mpn16 , respectively . in conclusion , although whole genome sequencing has become rapid and affordable to replace older typing methods in the future for either clinical strains or clinical specimens , mlva typing using the method by dgrange et  al . mlva typing was performed with good correlation in six international laboratories , indicating that  this methodology can be correctly performed on m.  pneumoniae at different locations . differences due to interpretation of fragment size , repeat sequence identification and repeat numbering led to inconsistencies in the final profiles assigned by laboratories . with users following the interpretation guidelines we provide , full interlaboratory strain comparison should be achieved .
typing of mycoplasma pneumoniae by multiple - locus variable - number tandem repeat analysis ( mlva ) is increasingly in use . 
 however , no specific internationally agreed guidance is available . 
 thirty m.  pneumoniae dna samples including serial dilutions of a type strain were sent to six international laboratories to perform mlva and results were compared . 
 good correlation was observed , indicating that this methodology can be robustly performed in multiple sites . 
 however , differences due to interpretation of fragment size , repeat sequence identification and repeat numbering led to inconsistency in the final profiles assigned by laboratories . 
 we propose guidelines for interpreting m.  pneumoniae mlva typing and assigning the number of repeats .