Molecular Diagnostic Approaches Molecular Diagnostic Approaches to Emerging Infectionsto Emerging Infections

三軍總醫院三軍總醫院 盧章智盧章智

WNVWNV

Are infectious diseases Are infectious diseases emerging more recently emerging more recently than before?than before?

Infectious Disease Mortality in Infectious Disease Mortality in the United States, 1980the United States, 1980--19961996

Source: JAMA 1996;275:189-193 and unpublished CDC data

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1990

1992

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1996

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美國醫學研究院（Institute of Medicine, IOM; 1992）：近年來（通常指近二十年內）出現於人類身上之新疫病，且其發生率呈現快速增加之趨勢，且在地理分布上之範圍擴張；且/或 增加或發展出新的抗藥性機制。

新興傳染病的分類

新興感染症之定義

舊病原→新微生物病原株種, 霍亂菌01/0/39在亞洲的流行舊病原→新宿主, 禽流感病毒H5N1在2004年越泰兩國造成人的新

型流感舊病原→復發流行, 台灣在1942~1943年曾有登革出血熱流行，

2002年台灣南部又有登革出血熱，六十年來未見最大流行

新病原→新病症, 新型冠狀病毒(SARS-CoV)→SARS舊病原→新病症, 漢他病毒在美國四角洲造成人的肺症候群(HPS)舊病原→新流行區, 西尼羅病毒在美洲擴散

1992年 美國紐約市--多重抗藥性結核菌1993年 美國南部四州--漢他肺症候群病毒1995年 英國--庫賈氏病傳染性變性蛋白質1996年 日本大阪地區-- O157型大腸桿菌1997年 香港--禽型流行性感冒病毒1999年 馬來西亞--立百病毒(Nipah Virus)1999年 美國紐約市--西尼羅病毒(West Nile Virus)2003年 全球SARS病毒

國際新興感染症 -近年的著名事件-

http://www.cdc.gov.tw/

台灣地區新興及再浮現感染症案例

萊姆病鉤端螺旋體病結核病漢他病毒肺症候群抗萬古黴素的金黃色葡萄球菌

93年12月29日公告公告公告新型流行性感冒為法定傳染病為法定傳染病

炭疽病HIV/AIDSSARS新型流感

目的賦予防治措施之法定效力，以使疫情來臨時，得以據法強制執行防治措施

公告內容疾病定義：採檢條件、疑似病例、確定病例流行疫情等級：0級、A1級、A2級、B級及C級

http://www.cdc.gov.tw/

Avian influenza A virus H9N2 was isolated from two children in Hong Kong in 1999.

Avian influenza H7N7 infected 89 persons during a simultaneous outbreak in poultry in the Netherlands in 2003.

The first outbreak of a highly pathogenic avian influenza (H5N1) in humans occurred in Hong Kong in 1997; 6 of 18 people with confirmed infection died.

Two cases of influenza A H5N1 occurred in Hong Kong in February 2003, followed by outbreaks in Vietnam and Thailand in January 2004.

Introduction

Apisarnthanarak A, Kitphati R, Thongphubeth K, Patoomanunt P, Anthanont P, Auwanit W, et al. Atypical avian influenza (H5N1). Emerg Infect Dis. 2004 Jul

Phylogenetic relationships among H5 hemagglutinin (HA) genes from H5N1 avian influenza viruses and their geographic distribution.

Evolution of H5N1 Avian Influenza Viruses in Asia. The World Health Organization Global Influenza Program Surveillance Network. Emerging Infectious Diseases Vol. 11, No. 10, October 2005

A variety of specimens from mammals and birds are suitable for the diagnosisof virus infections of the upper respiratory tract:

• nasal swab• throat swab• tracheal swab

Specimens should be collected and transported in a suitable transportmedium with antibiotics in ice or in liquid nitrogen.

流感快速檢驗試劑

以生研Seiken 試劑組為例。取含有病毒粒子之病毒液200uL 加入試劑組所附之檢體緩衝液中，混合均勻後用內附的濾膜蓋過濾。於檢測盤之A 與B 檢測孔中，分別滴入5滴檢體液，待檢體液完全被吸附後，再分別於A 檢測孔加入5 滴Influenza A抗體，而在B 檢測孔加入5 滴Influenza B 之抗體。待抗體完全被吸附後再加入5 滴清洗液，最後加入5 滴呈色液，靜置5-10 分鐘後進行結果判讀。

Specimens for diagnosis

Influenza Realtime PCR for H1 & H3, if rapid Influ A test is positive but H1 & H3 PCR are negative, then suspect Avian influ and perform H5-PCR

Primer and probe sequence:

H5F（H5 + 1456）：ACg TAT gAC TAT CCA CAA TAC TCA gH5R（H5 - 1685）：AgA CCA gCT ACC ATg ATT gCH5 probe：FAM-TCA ACA gTg gCg AgT TCC CTA gCA-TAMRADevelopment of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes.

Spackman E, Senne DA, Myers TJ, et. al. 2002. J Clin Microbiol. 40(9):3256-60.

Real time RT-PCR (ABI 7000 or Roche LightCycler )

RNA extraction

Qiagen RNAeasyTM Total RNA Isolation Kit (Catalog #74104):

Bacterial 16S Bacterial 16S rDNArDNAfound in all bacteriafound in all bacteriamutations at a slow constant ratemutations at a slow constant ratecontains extremely conserved regions contains extremely conserved regions of nucleotides alternating with regions of nucleotides alternating with regions that are variable between species but that are variable between species but constant within a speciesconstant within a species

SA 3992SE 3918L.mono 3566Entero 3464S.agal 3256S.pneu 3118S.pyogens 3116M.tub 2810S.typhi 2800SM 2790

100

100 200 300 400 500 600 700 800 900

200 300 400 500 600 700 800 900

E.coli 2790KP 2784Ps.aeru 2768E.cloaca 2764Proteus vu 2756Leg 2736N.men 2708

U1 : 5'-CCAGCAGCCGCGGTAATACG-3'U2 : 5'-TACGGCTACCTTGTTACGACTTC-3'

U1 U2

Universal (consensus) PCR

20721500

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M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

996

HistoryHistory2003.07.072003.07.07An 51An 51--year old Chinese female, year old Chinese female, housewifehousewife ..Lower abdominal pain and nausea. Lower abdominal pain and nausea. Admission diagnosis: Admission diagnosis:

HCV related cirrhosis of liver withHCV related cirrhosis of liver withacute exacerbationacute exacerbation

Laboratory dataLaboratory dataAn oral temperature of 36.4An oral temperature of 36.4℃℃ and no sign of trauma or other and no sign of trauma or other focus of infection was found . focus of infection was found . Leukocyte count 5.4 Leukocyte count 5.4 ××101033/uL, /uL, Hemoglobin level 10.0 g/dl, Hemoglobin level 10.0 g/dl, Platelet count 19 Platelet count 19 ××101033/uL/uL..

Serum albumin 2.5 g/dl,Serum albumin 2.5 g/dl,AST 79U/L, AST 79U/L, ALT 60U/L. ALT 60U/L. CultureCultureUrine culture No growth for 48 houUrine culture No growth for 48 hours.rs.Sputum culture Normal pharyngeal florSputum culture Normal pharyngeal flora.a.Blood culture Blood culture positivepositive..

Blood cultureBlood cultureThe aerobic blood culture positive.The aerobic blood culture positive.GramGram--positive positive coccuscoccus arranged in chains. arranged in chains. Slightly Slightly mucoidmucoid, beta, beta--hemolytic colonies after 24 hrs. hemolytic colonies after 24 hrs. CatalaseCatalase test (test (--) ) 、、PYR (+) PYR (+) 、、 BEM (BEM (--) ) LancefieldLancefield groups (A, B, C, D, F, or G) groups (A, B, C, D, F, or G) ：： ““none none ““VitekVitek 2 system (ID2 system (ID--GPI) GPI) ：："unidentified"unidentified““Sensitivity testSensitivity test

sensitive sensitive ：：penicillin penicillin 、、 erythromycinerythromycin、、ampicillinampicillin and and vancomycinvancomycin

intermediate intermediate ：：clindamycinclindamycin and and chloramphenicolchloramphenicol..

The hemolysis and colony morphology on 5% blood agar plate ( incubated at anaerobic)

Catalase(-) Catalase(+)

PYR(+) PYR(-)

Arginine(+) ； BEM(-) ； VP(-) ； Urea(-)

SXT (susceptible)

SXT

Bacitracin (susceptible)

B

Lancefield groups (A, B, C, D, F, or G) ： “none “

Forward: 5F/ gAA gAg TTT gAT cMT ggc Tc

Reverse: 809R/ gcg Tgg AcT Acc Agg gTA Tc

Sequencing primer: 531R/ TAc cgc ggc Tgc Tgg cA

5F 809R531RHypervariable regions

430 500267129

Total Amplified Section (804 bp)

Total gene: coding for 16S rRNA gene

A diagram of the 16S rRNA gene. Red boxes are hypervariable regions in the 5’region of the 16S rRNA gene.

Invasive infection of Streptococcus iniaein Taiwan

葉靜穎、孫俊仁、黃麗文、＊嚴助成、盧章智

三軍總醫院臨床病理科、＊腸胃科、國防醫學院病理學科

Colony morphologyGrowth ratePigmentation

Conventional method:Nitrate reductionNiacin3 days arylsafataseTween 80 hydrolysisSemi-quantitative catalase

16S rDNA sequencing:DNA extractionPCR amplicationPurification and quantification Sequecing PCRSequencing Sequence data analysis

Comparison and result interpretation

2 days 2-3 weeks

L-J medium growth,Acid Fast stain positive

Descriptive term Conventional identification No. ofisolates16S rDNA sequencing

identificationNo. of

isolates

group I

photochromogenic Mycobacterium kansasii 12 Mycobacterium kansasii 12

slow growers Mycobacterium marinum 2 Mycobacterium marinum 2Mycobacterium asiaticum 1 Mycobacterium asiaticum 1

group II

scotochromogenic Mycobacterium gordonae 40 Mycobacterium gordonae 40

slow growers Mycobacterium scrofulaceum 5 Mycobacterium scrofulaceum 5Mycobacterium xenopi 1 Mycobacterium xenopi 1

group III

nonphotochromogenic MAC 60 M. avium 21

slow growers M. intracellulare 30M. lentiflavum 9

M. terrae complex 9 M.terrae 5

M. nonchromogenicum 4

M. celatum 1 M. celatum 1

M. tuberculosis 6 M. tuberculosis complex 6

group IV

rapid growers Mycobacterium chelonae 51 M. chelonae-abscessus 46species group M. immunogenum 4

M. mucogenicum 1

Mycobacterium fortuitum 32 Mycobacterium fortuitum 32

M. smegmatis 1 M. smegmatis 1

Total 221 221

StepStep Total cost of Total cost of materialsmaterials Labor (hr)Labor (hr)

lysatelysate preparation and DNA extractionpreparation and DNA extraction 55 2.52.5

PCR PCR amplicationamplication and Electrophoresisand Electrophoresis 1515 22

PCR product purification and quantification PCR product purification and quantification 1515 0.50.5

SequecingSequecing PCR (PCR (BigDyeBigDye Terminator Sequencing Kit)Terminator Sequencing Kit) 3535 22

Sequencing (ABI Prism 310 Genetic Analyzer)Sequencing (ABI Prism 310 Genetic Analyzer) 3030 2.52.5

Sequence data analysis (NCBI or RIDOM blast)Sequence data analysis (NCBI or RIDOM blast) 00 0.50.5

Total Total 100100 1010

Bacillary Angiomatosis

Caused by Bartonella henselae

Caused by gram-positive actinomycete, Tropheryma whippelii

Representational Difference Representational Difference Analysis (RDA)Analysis (RDA)

RDA was first described by Drs. Nikolai RDA was first described by Drs. Nikolai LisitsynLisitsyn, , NatalyaNatalyaLisitsynLisitsyn, and Michael , and Michael WiglerWigler of Cold Spring Harbor in of Cold Spring Harbor in ScienceScience in February 1993 (Vol. 259, p. 946). in February 1993 (Vol. 259, p. 946). By comparing DNA from diseased and normal cells from a By comparing DNA from diseased and normal cells from a person, the method can identify DNA sequences that differ person, the method can identify DNA sequences that differ between diseased and normal cells from the same person. between diseased and normal cells from the same person. Also, it can assess genetic changes that occur during Also, it can assess genetic changes that occur during development and detect deletions, rearrangements, or development and detect deletions, rearrangements, or extraneous viral DNA sequences. RDA enriches for unique extraneous viral DNA sequences. RDA enriches for unique DNA sequences from one of the genomes by removing DNA sequences from one of the genomes by removing shared sequences. shared sequences. It takes advantage of It takes advantage of PCRPCR to amplify those distinct regions. to amplify those distinct regions.

Linker

A

B

A

B

Representational difference analysis

Representational Difference Analysis

mRNA cDNA

GATCCTAG

Ligate R adaptersDpnII

PCR with R primers to generate amplicons, digest with DpnII

Tester(EML)

Driver(EPRO)

Ligate 12/24J adaptersto tester only Mix, melt, hybridize

PCR with J primers

Exponential amplification

Linear amplification

No amplification

Retriction fragments

Digested KS diseased cells hybridized with linkers

Hybridized with digested non-diseased cells

(Subtractive hybridizatiion)

Normal DNA without primer to normal DNA without primer

Normal DNA with primer from diseased tissue to normal DNA without primer

Unique DNA with primer will only re-anneal to unique DNA with primer from diseased tissue

Thanks for your attention !

Molecular Diagnostic Approaches to Emerging InfectionsWNV Infectious Disease Mortality in the United States, 1980-1996Bacterial 16S rDNAHistoryLaboratory dataBlood cultureRepresentational Difference Analysis (RDA)