結核および多剤耐性結核の診断のためのMODSの方法

For every three people in the world, one is infected With tuberculosis. Diagnosing active tuberculosis prevents new infections because patients with active tuberculosis will go on to infect between 10 and 15 other people per year until the time when they are either treated Or die. Mods is an acronym That we use to avoid having to say microscopic observation, drug susceptibility assay, and the reason that it's called that will become apparent as the methodology is explained.

In the short video, we will outline the mods methodology for the rapid inexpensive diagnosis of tuberculosis and multi-drug resistant tuberculosis. The video will set out the methodology step by step. We'll discuss the biosafety implications of working with liquid culture, and we'll also explain some of the important quality assurance issues that are required for use of this methodology.

The mods assay was developed at Kayana University in Lima, Peru in the laboratory of Bob Gilman. Following observations initially made by loose cavi. LOOSE was trying to identify rapid ways of detecting mycobacterium tuberculosis in cultures using colormetric indicators.

But she noted that the observation of those cultures under the microscope revealed the presence of growth of microbacteria several days before the Colormetric indicators changed color, and it was based on that observation that the MOD's assay was developed. MOD's assay was developed in a resource limited setting and is targeted at resource limited settings, therefore just under 3 per test for liquid culture based detection of TB and multi-drug resistance detection. It's extremely appealing and for the first time brings that sort of diagnostic capacity to places where resources are limited.

So the mods assay is based on three central principles. The first is that mycobacterium tuberculosis grows in liquid media much more rapidly than it does in solid media. This is a fact that's been known for many years.

Second is that if we observe those liquid media under the microscope, we can detect the growth of myb tuberculosis much more rapidly than waiting for the macroscopic appearance of colonies on solid media. And indeed, that growth is entirely characteristic such that we can distinguish M tuberculosis in liquid media from bacteria or fungal contamination, or indeed from the growth of atypical nontuberculous mycobacteria that might be present. Finally, and this is perhaps the most exciting thing about the mods assay, is that we can incorporate the drugs is rifampicin and therefore multi-drug resistance directly from sputum.

By using those drugs at the outset of the assay, we can detect both tuberculosis and at the same time whether or not the isolate is resistant to those two drugs. And this obviates the need to subculture and create an indirect drug susceptibility test. So now we're just gonna outline the equipment that is required to do the mods assay.

A fridge is needed in order to store prepared media. A balance is required to weigh the is and Rifampin. A vortex is needed for mixing of sputum.

With the decontamination solution, centrifuge is required for concentrating the decontaminated sputum sample, and this should be capable of achieving 3000 G.The centrifuge need not be refrigerated, but it is absolutely essential that it is bios safe and to be bios safe. For mycobacterial work that requires that the buckets of the centrifuge can be sealed with lockable lids. An incubator that can achieve 37 and maintain 37 degrees centigrade is required.

This need not be CO2 enriched. An inverted microscope is required to read the mods plates, and this should have objectives of four x and 10 x and autoclave is needed to sterilize media and also for disposal of microbacterial culture, waste micros are required for aliquot in media, a biological safety cabinet. Class two is required for procedures involving manipulation of sputum samples and inoculation of plates.

The reagents required to perform the mods asay are as follows, Middlebrook seven H nine media cascione and glycerol Panta, which is the antibiotic supplement and OADC. The nutritional supplement stocks of the antibiotics is and Rifampin dimethyl sulfoxide, DMSO for preparation of rifampicin, sodium hydroxide, sodium citrate, and nyl cysteine for sputum decontamination di, sodium phosphate and mono potassium phosphate for preparation of the phosphate buffer, 10%sodium hypochlorite for disposal of infected waste and disinfectant for cleaning work surfaces. The following plastics and consumables are also required sterile glass tubes 0.2 micrometer filters for aqueous solvents and 0.2 micrometer filters for organic solvents, micro centrifuge tubes, 15 mil centrifuge tubes, sterile 50 milliliter, 10 milliliter serological peppes past peppes sterile micropipet tips 24 well tissue culture type plates and sealable Ziploc type ine bags is which the 24 well plates are placed after the sputum sample is inoculated into the media.

There are four groups Of stock solutions that can be prepared in advance. The phosphate buffer Solution is made of a combination of sodium phosphate, diba, and potassium phosphate mono basic solutions adjusted to a pH of 6.8 plus or minus 0.2. This solution is autoclaved at 121 to 124 degrees centigrade for 15 minutes to sterilize, and an aliquot is cultured on nutrient agar to confirm sterility.

This may be stored in the refrigerator at two to eight degrees centigrade for up to one month. Each sputum sample requires 10 milliliters of Phosphate buffer solution. The stock solution For sputum decontamination is a sodium hydroxide sodium citrate combination.

These solutions are combined in equal volumes and autoclave at 121 to 124 degrees centigrade for 15 minutes and may also be stored in the refrigerator at two to eight degrees centigrade for up to one month. Two milliliters of this solution is required for each sputum sample. The basic culture medium is composed of Middlebrook seven H nine with cascione and glycerol.

The seven H nine powder is dissolved in sterile distilled water containing glycerol and cascione and mixed with constant agitation until completely dissolved. This media is autoclave at 121 to 124 degrees centigrade for 15 minutes, and after cooling is divided into 4.5 milliliter quats in sterile 16 by 100 millimeter glass tubes. The media is incubated at 37 degrees for 48 hours to verify sterility, which is indicated by a lack of turbidity after this time and may be stored at two to eight degrees centigrade with the cap tightly closed for up to one month.

Each sputum sample requires one tube containing 4.5 milliliters of this culture. Medium antibiotic stock solutions Are prepared as follows is, is dissolve completely in sterile distilled water and filtered with a 0.2 micrometer syringe filter for aqueous solvents. 20 microliter aliquots can then be stored in sterile micro centrifugation tubes at minus 20 degrees centigrade for up to six months each 20 microliter aliquot is sufficient for up to a hundred samples.

Rifampin stock solutions of eight milligrams per mil are prepared by dissolving rifampicin in DMSO and sterile distilled water. This mixture is filtered with a 0.2 micrometer syringe filter for organic solvents and the solution can be stored in 20 microliter aliquots in sterile micro centrifuge tubes at minus 20 degrees centigrade for up to six months each 20 microliter aliquot is sufficient For up to 100 samples. So now we come to the part of the methodology itself and the three steps that you need to take on the day of procedure.

This is converting the stock solutions into the working solutions. First we have the decontamination solution, then we have the media solution. And finally, the working solutions of the drugs isen and Rifampin.

The decontamination method, which is used for the mods methodology, is the standardized sodium hydroxide nalk decontamination methodology. The nalk is a mucolytic, which helps to digest the sputum and allow the sodium hydroxide to do the decontamination. For this, we need our sodium hydroxide sodium citrate stock solution and 0.1 grams of NA is added to 20 mls of that solution.

Any solution that is not used on the day of preparation has to be discarded. The final preparation of the culture medium should also only be done on the day of use. Stored seven H nine media is combined with OADC and panta 4.5 milliliters of the media is combined with 0.5 milliliters of OADC and 0.1 milliliters of Panta in each tube.

This is sufficient for one sample. Working solutions of each drug are prepared on the day of use and any solutions that are not used should be discarded because activity cannot be guaranteed stored aliquots are diluted in 24. Well tissue culture plates serially with media seven H nine oh A DC such that the final well concentration that is achieved after the sputum sample with media has been added is 0.4 micrograms per mil foric acid and one microgram per mil for Rifampin.

On now to the Decontamination procedure, two mils of sputum sample is placed into a conical based Falcon tube and two mils of our sodium hydroxide milk Decontaminate solution is then added. The lid is replaced and tightly screwed on after which the suspension is vortex and left to stand for 15 minutes during which time the decontamination procedure takes place. After this time, 14 milliliters of phosphate buffer solution is added.

The lid is replaced and screwed tight, and the tube is placed into the centrifuge after 50 minutes at 3000 G, the S supernatant is decanted and the pellet that remains is the sample which we wish to plate out using the 5.1 milliliter tubes of seven H nine OEDC panter. Our pellet is resuspended to a volume of approximately two milliliters. Half of this resuspended sample is stored as a backup in a micro centrifuge tube.

This is then available in case our culture becomes contaminated and we need to plate out the sample again. The other milliliter is added to the remaining seven H nine oh a DC panter that is in our glass tube, and this is our sample that is ready for plating out. 900 microliters of this suspension of media and sample is placed in each of four wells in a column of a 24 well plate.

The remaining wells of the 24 well plate are filled with subsequent samples such that five samples fill a 24 well plate, one for each of five columns, and the sixth column is left without a sample. The sixth column serves as a negative control into this column. It's placed all the media constituents without a sputum sample and clearly any growth in this column indicates that we have a problem with cross contamination and the whole plate needs to be discarded.

Every plate that we run requires a column of negative control. So now we have our plate containing all of our sample and media. We have to add our antibiotic suspensions in a separate plate.

We've prepared our dilutions as previously described of Rifampin and Isid, and with a four channel multi pet, we collect 100 microliters from each of these wells and dispense 100 microliters of seven H nine OADC to each of two control wells and 100 microliters of Rifampin and 100 microliters of isid to each of the two drug containing wells. Once the plate is complete, the lid is replaced and it's placed inside. A Ziploc bag from this point on the bag isn't opened again, and therefore, any cultures that grow in liquid media never have the opportunity to leave the bag.

All of the readings of the plate from now on in are done within the bag, and the bag is placed in an autoclave bag and discarded at the end of the culture. And in this way, we avoid manipulation of highly concentrated suspensions of mycobacteria. We'll just say a few words now about quality control.

There are inbuilt quality controls in the mods methodology, which are essential for the proper use of the assay. First of all, there are negative controls and these are run on every single plate. These have been mentioned previously, but in summary, we have a column that contains all the media constituents without the presence of a sputum sample alongside five other sputum samples on a plate, the wells in column should be examined just as any other wells and the presence of any growth in these wells indicates the cross-contamination has taken place and that none of the results on the plate can therefore be trusted.

The plate must therefore be discarded and all the samples must be replated. In addition, there are internal controls that are positive controls. These are run on a separate plate, and this plate is prepared after all the other sample plates have been prepared at the end of the day to minimize the risk of cross-contamination within the laboratory.

On this separate plate, we run one fully susceptible strain of mycobacterium tuberculosis and a strain that is resistant to isid and rifampicin. If there are concerns about running a multi-drug resistant strain on a plate, a rifampin mono resistant strain may be run without is mono resistant strain in place of these two susceptible and MDR strains. The purpose of these strains is to demonstrate not only that the media support growth, therefore all of our positive controls should have growth in the drug free wealth, but also that our drug concentrations are correct and therefore the susceptible positive controls should not grow in the presence of is and Rifampin and the resistant positive controls should obviously grow through the concentrations of Rifampin and is which are present in the plate.

Okay, so now on to plate reading. If we regard the day of plating out of our samplers, day zero plate reading starts on day five. Prior to that, mycobacterial growth is very rarely seen.

Plates are examined under a inverted light microscope. That is to say that the plates are examined from below upwards, and this allows us to see the cords very clearly attempting to visualize the cultures from above is hampered by condensation on the lid of plates, and also because the focal length is is not adequate of a standard microscope. Microscopic examination is generally undertaken with the 10 x objective positive cultures regarded as one in which there are at least two colony forming units in both drug-free wells.

The presence of contamination is readily recognized because the media becomes cloudy and by day five there is generally overgrowth of most of the well. In contrast, mycobacterial growth does not lead to a cloudiness of the well initial growth of mycobacteria form small comma like forms, which steadily progress to form characteristic chords or tangles. Within two weeks, a strongly positive growth will have formed conglomerations of mycobacteria with cords only really visible at the extremities.

The vast majority of cultures that are going to become positive are positive by day 14, and therefore we recommend daily readings from day five to day 14 and thereafter readings every two or three days according to laboratory workload on the day that a culture is clearly positive in the two drug-free wells, the drug containing wells should be read. Any growth in the presence of either drug indicates resistance to that drug. It's very important to note that if the region of the drug containing wells is done more than one week after a positive culture is noted in the drug free wells, this may not represent resistance as occasional breakthrough growth is seen.

Readings can stop at day 21 if a culture is negative, as positive cultures after this time are never encountered in patients not receiving therapy On completion of 21 days of incubation, cultures may be discarded. Cultures remain within their Ziploc bags and are placed in autoclave bags and autoclave at 121 to 124 degrees centigrade for 45 to 60 minutes after which they may be safely discarded. I'm just gonna say a few words now about biosafety and the mods assay as we've been rolling mods out not only in Peru but also in settings in Africa and Asia.

A recurrent question arises about the security and the biosafety of using microbacterial cultures in liquid media. This is an understandable fear because obviously liquids can be spilled and solid media don't carry that risk, and also there's a risk of aerosolization when we're dealing with suspensions of mycobacteria. It's actually because of these very reasons that we believe that the mod methodology is a safer methodology than any methodology that requires indirect drug susceptibility testing.

The reason for this is that for indirect susceptibility testing, a suspension of a known concentration of microbacteria is prepared. This therefore involves the manipulation of highly concentrated solutions of microbacteria with all the attendant risks of spillage and aerosolization. In contrast, the mods methodology, as you will now have seen, simply involves the inoculation of a sputum sample into our plate, after which the plate is sealed within a plastic bag.

This bag is never opened again, and therefore we don't have to deal with these highly concentrated suspensions. This isn't to say that we believe that the mod methodology is completely safe handling mycobacteria always carries a risk. However, data from Korea published in 2006 seemed to have vindicate our position that mods is no more dangerous than plating out any other culture.

This was a study that was undertaken in laboratory workers, which showed that the occupational risks to laboratory workers who plated out cultures and had no responsibility for drug susceptibility testing was no greater than that for those who are doing sputum smear microscopy. In contrast, those who are handling suspensions of mycobacteria and doing drug susceptibility testing have much higher rates of occupational risk for tuberculosis. Clearly, it's of paramount importance that all labor laboratory staff are protected as far as they possibly can, and we therefore strongly recommend that none of these procedures be carried out without appropriate personal respiratory protection by which we mean N95 standard masks.

In addition, a class two biological safety cabinet or hood is required in which all exhausted areas filtered through HEPA filters. Finally, a simple mechanism that's very important in avoiding turbulence of airflow within a laboratory is a lock on the laboratory door to stop entry and exit of personnel while samples are being processed. This reduces the risk of transmission within the laboratory, So I hope that that's Given you a, a good overview of what the mods methodology involves and and how useful it might be in settings where resources are limited, but high quality TB and MDR TB diagnostics are urgently required.

For more information, you can visit our website, mods peru. org, where you'll find our detailed standard operating procedures, the user guide to mods. In addition to SOPs for the processing of extra pulmonary samples, we also have a photo library of mycobacterium and tuberculosis, other mycobacteria and bacterial and fungal contamination, and you can also find our quality assurance strategy and the procedure that we recommend for accreditation of laboratories to start using mods.

Finally, there is a frequently asked questions sheet, and we welcome any feedback from users who are currently using mods as this is an iterative methodology. As we explained earlier, it's a non-proprietary test and we're always interested in improvements that other laboratories have managed to make.