WEIRD BUGS IN YOUR BRAIN?

Tape­worms and mould fungi could at­tack your brain and make you fa­tally ill. Of­ten, doc­tors do not iden­tify the at­tack­ers and are forced to "fire at ran­dom". How­ever, a new, ground-break­ing ge­netic method can now re­veal the lethal or­gan­isms, be­fore it is t

Science Illustrated - - CONTENTS -

Feel­ing a bit off? It mightn’t be your at­ti­tude, it might just be the baby tape­worm lodged in your ac­tual brain. Yes, it’s gross.

Doc­tors are un­able to find out what is wrong with the 14-year-old girl. She is ad­mit­ted to hospi­tal in the sum­mer of 2015 with se­vere fever and headache. Af­ter a few days, she be­comes deliri­ous, only speaks in brief sen­tences of no more than three words, and does not re­act, when spo­ken to. The symp­toms in­di­cate a type of menin­gi­tis, which could be caused by 100+ dif­fer­ent path­o­genic or­gan­isms, that all re­quire dif­fer­ent treat­ments. Sci­en­tists must find the cause to be able to give the girl the right drug, and so, they ini­ti­ate a series of tests.

Each test is cus­tom­ized to re­veal the pres­ence of one sin­gle type of or­gan­ism, and doc­tors test for one af­ter the other, hop­ing to fi­nally get a pos­i­tive re­sult. How­ever, all test re­sults are neg­a­tive. The girl is in­fected with some­thing that only rarely at­tacks the brain. There might be a test for the or­gan­ism, but doc­tors have no idea what to look for. Af­ter 19 days, they give up and de­cide to send a sam­ple of the girl’s cere­brospinal fluid to bio­chemist Joe DeRisi’s re­search lab­o­ra­tory at the Univer­sity of Cal­i­for­nia, San Fran­cisco, USA, where a new test is in the process of rev­o­lu­tion­iz­ing med­i­cal science.

Test iden­ti­fies mi­crobes

Ac­cord­ing to DeRisi, it is “the mother of all tests”. Un­like the tests that doc­tors use, it is not meant to show the pres­ence of one spe­cific or­gan­ism. In­stead, it scans the sam­ple to find ev­i­dence of ev­ery­thing that should not be there, and it does not dis­tin­guish be­tween bac­te­ria, fungi, an­i­mals, or other par­a­sites.

The most im­por­tant thing about the test is, how­ever, that it does not only sound the alarm, when it spots an unau­tho­rised or­gan­ism in the sam­ple. It im­me­di­ately names the in­truder, al­low­ing the doc­tor to make a di­ag­no­sis and start the right treat­ment. It cor­re­sponds to a

bur­glar alarm, which im­me­di­ately iden­ti­fies the bur­glar and sends his name and ad­dress di­rectly to the po­lice.

As the girl’s symp­toms grad­u­ally get worse, in­clud­ing cramps, stiff­ness, and trem­bling arms and legs, Joe DeRisi and his col­league, Michael Wil­son, be­gin their analy­ses. The cere­brospinal fluid con­tains salts, su­gar, hor­mones, fat, and a few pro­teins and white blood cells, but the two sci­en­tists are look­ing for some­thing com­pletely dif­fer­ent. They are in­ter­ested in ev­i­dence of genes that have es­caped from cells in the young girl’s brain.

When brain cells die, they are bro­ken down, and the resid­ual prod­ucts are drained from the brain with the cere­brospinal fluid. So, this is where to find ev­i­dence of the cells’ ge­netic ma­te­rial – not only of genes from the girl her­self, but also from any con­ta­gious or­gan­ism which might have in­vaded her brain. And that is the very "alien" genes that DeRisi and Wil­son are look­ing for.

Sci­en­tists helped 300 pa­tients

With DeRisi’s new test, doc­tors no longer need to try to guess what caused the in­fec­tion and make a test for ev­ery guess. Valu­able time could be wasted in this way, and like in the case of the 14-year-old girl, there is a risk that the many tests will never lead to a pos­i­tive re­sult. That is why in more than half of all cases of menin­gi­tis or cere­brospinal menin­gi­tis, doc­tors never man­age to name the path­o­genic or­gan­ism.

Iden­ti­fy­ing the cause of the dis­ease is key to the pa­tients’ health, as whereas brain in­fec­tions caused by bac­te­ria can be com­bated with an­tibi­otics, th­ese drugs are in­ef­fec­tive in con­nec­tion with virus, and fungi­cides are no good, when it comes to menin­gi­tis which is caused by a par­a­sitic worm.

In 2013, DeRisi and Wil­son first used the new test to iden­tify an or­gan­ism that had in­fected the brain of a pa­tient who doc­tors were un­able to di­ag­nose in the nor­mal way. Since then, the two sci­en­tists and sev­eral of their col­leagues have helped more than 300 pa­tients and their doc­tors es­tab­lish dif­fi­cult di­ag­noses.

With the as­sis­tance of physi­cian Charles Chiu, who is also from the Univer­sity of Cal­i­for­nia, San Fran­cisco, and oth­ers, they have sys­tem­ized the anal­y­sis method, so it is more or less au­to­matic. And since July 2017, Amer­i­can doc­tors have been able to or­der an anal­y­sis for their pa­tients over the In­ter­net at a price of some $3,000. The doc­tor only needs to take a sam­ple from the pa­tient, freeze it, and send it to San Fran­cisco. And only 72 hours af­ter the sam­ple has ar­rived, the doc­tor re­ceives the name of the or­gan­ism.

Genome ed­i­tor ti­dies up DNA

In the lab, DeRisi and his team are now analysing the sam­ple from the 14-year-old girl. The small quan­tity of liq­uid con­tains mil­lions of DNA se­quences, but the sci­en­tists are only in­ter­ested in a small hand­ful of them – the ones com­ing from the path­o­genic or­gan­ism. So, the sci­en­tists aim to screen out the girl’s own DNA se­quences, and that is done by means of the new CRISPR genome edit­ing tech­nol­ogy.

CRISPR is like a fo­cused molec­u­lar edit­ing tool that can be de­signed to cut up spe­cific DNA se­quences. DeRisi’s team has cho­sen to cut 266 dif­fer­ent se­quences, which are very com­mon in peo­ple, but rarely ob­served in other or­gan­isms. All gene frag­ments with th­ese typ­i­cally hu­man se­quences are cut up and screened out dur­ing the very first steps of the process. So, a large quan­tity of ge­netic "noise" is re­moved from the sam­ple, and the alien gene frag­ments stand out more clearly. Ac­cord­ing to DeRisi’s ex­per­i­ment, this process re­moves about one third of the un­wanted DNA se­quences from the spec­i­men, mak­ing the test four times bet­ter at iden­ti­fy­ing the alien or­gan­ism’s DNA.

In spite of the clear-out, the sam­ple still con­tains al­most eight mil­lion DNA se­quences, of which more than 99.99 % be­long to the girl her­self. The sci­en­tists now need to read ev­ery sin­gle DNA se­quence very ac­cu­rately to find the small hand­ful of se­quences from the path­o­genic or­gan­ism. Pre­vi­ously, this would have re­quired that the DNA se­quences be read one at a time via a man­ual, time-con­sum­ing process. But a new method known as metage­nomic nex­tgen­er­a­tion se­quenc­ing (mNGS) makes it pos­si­ble to se­quence about 20 mil­lion DNA se­quences at the same time and make the com­puter reg­is­ter ev­ery sin­gle se­quence.

In short, the se­quenc­ing takes place by sci­en­tists peel­ing one strand off all DNA se­quences, caus­ing en­zymes to start recre­at­ing the miss­ing strands. Sub­se­quently, a cam­era and a com­puter can mon­i­tor the re­cre­ation of ev­ery sin­gle strand. The equip­ment can de­ter­mine which DNA bases are added and in which or­der. The re­sult is that the com­puter reg­is­ters the se­quence of DNA bases of ev­ery sin­gle DNA strand.

DeRisi’s team now has a vast quan­tity of data – mil­lions of se­quences con­sist­ing of the let­ters A, T, C, and G. The next step is an­other round of clear-out to re­move the re­main­ing hu­man genes. By means of so­phis­ti­cated al­go­rithms, a com­puter fil­ters mil­lions of se­quences per hour. It com­pares the se­quences to huge data­bases of hu­man genes, al­low­ing it to spot the girl’s DNA and fil­ter it out. Fi­nally, the sci­en­tists only have a small hand­ful of se­quences left. They are com­pared to known se­quences from thou­sands of or­gan­isms, un­til DeRisi has a short, but com­plete list of all the life forms that have left ev­i­dence in the girl’s brain.

Mos­quito car­ried rare dis­ease

The list con­tains four or­gan­isms: a virus that only at­tacks to­bacco plants, a blue-green alga, a soil bac­terium, and the West Nile virus. The three first ones are harm­less and very prob­a­bly did not cause the in­fec­tion in the girl’s brain. The fourth one, the West Nile virus, is very dif­fer­ent. It is closely re­lated to the Zika virus, dengue virus, and yel­low fever virus, and it of­ten at­tacks hu­mans. Nor­mally, the West Nile virus only causes flu-like symp­toms, but in very rare cases, the virus is able to pen­e­trate the brain’s pro­tec­tive bar­ri­ers and at­tack brain cells.

DeRisi’s team im­me­di­ately con­tact the girl’s doc­tors. They make an old-fash­ioned, tar­geted test, which is specif­i­cally de­signed to iden­tify the pres­ence of West Nile virus in the girl. The test is pos­i­tive. DeRisi’s ground-break­ing method has once again "hit the mark", and ev­ery­thing be­gins to make sense. Two days be­fore the girl's first symp­toms ap­peared, she was at a sum­mer camp by a small lake in a na­tional park out­side Los An­ge­les. And when she came to the hospi­tal, she had a large mos­quito bite on her leg. None of the doc­tors paid any at­ten­tion to it at the time, but the virus prob­a­bly en­tered her body via an in­fected mos­quito.

To­day, there is no ef­fi­cient treat­ment against the West Nile virus, but the girl’s dis­ease has sta­bi­lized. She has gone back to school, and she is do­ing well – but she still has prob­lems with speech and with her bal­ance. Fu­ture drugs might en­sure that she will get com­pletely rid of the alien or­gan­ism. How­ever, DeRisi’s di­ag­no­sis al­ready plays an im­por­tant role for her, pre­vent­ing that, due to ig­no­rance, doc­tors would treat her with po­ten­tially haz­ardous drugs that have no ef­fect on the dis­ease.

Be­fore the di­ag­no­sis, doc­tors had treated the girl with high doses of at least five dif­fer­ent types of an­tibi­otics and drugs against her­pes and Ep­stein-Barr virus. All the drugs in­volved a long series of po­ten­tial side ef­fects, but none of them were ef­fi­cient against West Nile virus. If DeRisi’s test had been as eas­ily ac­ces­si­ble to doc­tors as it is now, the di­ag­no­sis could have been made sev­eral weeks pre­vi­ously – and the

girl's chances of a quick re­cov­ery would have been im­proved.

Test also re­veals can­cer

So far, doc­tors are only al­lowed to use the test to an­a­lyse cere­brospinal fluid, so un­til fur­ther no­tice, it has only been used to di­ag­nose brain in­fec­tions. How­ever, sci­en­tists through­out the world are strug­gling to show that the method can be used in con­nec­tion with a long series of other dis­eases. Over the past five years, the an­nual num­ber of sci­en­tific ar­ti­cles about the sub­ject has been quin­tu­pled.

Ac­cord­ing to plan, doc­tors will also be able to have blood, tis­sue, urine, fae­ces, and slime sam­ples from the res­pi­ra­tory or­gans an­a­lysed, mean­ing that in a mat­ter of a few days, they will be able to find the cause of lethal cases of pneu­mo­nia or blood poi­son­ing, which could both be caused by very dif­fer­ent types of or­gan­isms.

DeRisi’s team has em­barked on a new project in­volv­ing eye in­fec­tions, which are also dif­fi­cult to di­ag­nose in the tra­di­tional way. One of the pa­tients who the sci­en­tists have ex­am­ined had an eye in­fec­tion for 16 years and had been checked by sev­eral doc­tors with­out an ac­cu­rate di­ag­no­sis be­ing es­tab­lished. The two sci­en­tists from San Fran­cisco took a sam­ple from the pa­tient’s eye­ball, and out of a to­tal of 12,111,540 gene frag­ments, 10 turned out to come from the rubella virus, that nor­mally causes measles.

How­ever, the method is by no means lim­ited to in­fec­tious dis­eases. It can also be used to re­veal dis­eases caused by mu­ta­tions in the pa­tient’s own genes. In prin­ci­ple, sci­en­tists use the same method, but in­stead of com­par­ing gene frag­ments in the sam­ple to DNA se­quences from alien or­gan­isms, they com­pare them to known, path­o­genic mu­ta­tions. In 2016, sci­en­tist David Dy­ment used the method to re­veal the ge­netic cause of a num­ber of oth­er­wise in­ex­pli­ca­ble dis­eases in eight new­born chil­dren.

The most harm­ful ge­netic dis­ease of all, can­cer, is also un­der fire. The dis­ease might de­velop as a re­sult of in­her­ited or new mu­ta­tions, and pre­vi­ously, doc­tors have been forced to go through the pa­tient’s genes one af­ter the other to find the mu­ta­tions. The new method scans thou­sands of genes at a time, and sci­en­tists are al­ready well on their way to us­ing DeRisi's method to make quick di­ag­noses of lung, prostate, and birth­mark can­cer.

STEVE BABULJAK/UCSF

Bio­chemist Joe DeRisi’s team has de­vel­oped a new method that can re­veal the iden­tity of all the or­gan­isms that have left ev­i­dence in a pa­tient’s brain.

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