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Referatbaru

  1. 1. DEMAM TIFOID<br />Aspek patogenesis, gejala klinis dan pemeriksaan laboratorium<br />Ferdy/ Jusak Nugraha<br />
  2. 2. RALAT<br />Judul tertulis: aspek patogenesis, klinis dan pemeriksaan laboratorium, seharusnya: aspek patogenesis, gejala klinis dan pemeriksaan laboratorium<br />Hal 5 kata pertama tertulis: kedua lipid, seharusnya: lipid<br />Hal 10 baris 1 tertulis endotil, seharusnya endotel<br />Hal 11 paragraf 1 baris 7 tertulis: independent, seharusnya dependent<br />Hal 25 baris 8 tertulis ynga, seharusnya yang<br />
  3. 3. Pendahuluan<br />Depkes1997: 350-810 kasus/ 100.000 penduduk /tahun<br />17 juta kasus baru <br /> 60.000 kematian<br />RSUD dr. Soetomo 1996-2000: dirawat 1563 penderita, angka kematian 1,09 % <br />♦ Strongly endemic♦ Endemic♦ Sporadic cases<br />Gambar 1. Insiden demam tifoid (sumber:www.wikipedia.org)<br />3<br />
  4. 4. Pendahuluan<br />batang, Gram negatif, berflagela, tidak membentuk spora dan bersifat fakultatif anaerob<br />familiEnterobacteriaceagenus Salmonellae<br />S.Typhi<br />tidak memetabolisme sukrosa dan laktosa<br />memetabolisme glukosa, maltosa dan manitol <br />4<br />
  5. 5. > 2000 varian<br />Berdasarkan jenis antigen:<br /><ul><li> O (somatik)
  6. 6. H (flagella)
  7. 7. Vi (kapsular)
  8. 8. Outer membrane protein (OMP)
  9. 9. Heat shock Protein (HSP)</li></ul>Klasifikasi<br />5<br />
  10. 10. Gambar 2. Dinding sel Salmonella (Sumber: Lim 2010)<br />6<br />
  11. 11. Serotipe (serovar)<br />Spesies dicetak miring<br />Serovar tidak dicetak miring dan mulai dengan huruf pertama kapital<br />Contoh: Salmonella enterica serovar Typhi. <br />Serovar dapat digunakan tanpa nama spesies, misalnya: Salmonella Typhi (S. Typhi)<br />Nomenklatur<br />7<br />
  12. 12. Tabel 1: Struktur antigen dari beberapa Salmonella umum<br />Sumber: WHO 2003<br />8<br />
  13. 13. Patogenesis<br />9<br />S.Typhi<br />S.Typhi<br />Gambar 3. Gambaran skematis infeksi S.Typhi (Sumber:Everest P 2010)<br />
  14. 14. Patogenesis<br />10<br />Gambar 3. Gambaran skematis infeksi S.Typhi (Sumber:Monack DM 2004)<br />
  15. 15. Klasik 4 stadium:<br />Minggu pertama: demam perlahan, bradikardia relatif, malaise, sakit kepala, hidung berdarah, nyeri perut, leukopenia, eosinopenia, limfositosis relatif<br />Minggu kedua: demam “nervous fever“, Rose spots, diare, konstipasi, hepatosplenomegali, peningkatan transaminase. <br />Gambaran klinis<br />pendahuluan<br />11<br />11<br />
  16. 16. Minggu ketiga: perdarahan intestinal, perforasi intestinal, septikemia, peritonitis difus, ensefalitis, abses metastatik, kolesistitis, endokarditis, osteitis<br />Akhir minggu ketiga: defervescene, karier<br />Klinis<br />pendahuluan<br />12<br />12<br />
  17. 17. Penting : sarana laboratorium <br />Gejala klinis sulit<br />Diagnosis<br />Isolasi kuman<br />1<br />Imunoasai <br />2<br />Pelacakan DNA Salmonella Typhi<br />3<br />13<br />
  18. 18. 1. Kultur darah<br />- baku emas<br />- positif > 80% pada minggu I<br />Kegagalan: <br />keterbatasan media, penggunaan antibiotika, jumlah bakteri minimal, volume spesimen tidak mencukupi dan waktu pengambilan spesimen yang tidak tepat<br />Pemeriksaan mikrobiologi<br />pendahuluan<br />14<br />14<br />
  19. 19. 2. Kultur tinja<br /><ul><li>daerah endemis: tidak memastikan diagnosis
  20. 20. daerah non endemis: membantu diagnosis demam tifoid</li></ul>3. Kultur urine<br /><ul><li> positif sekitar 25% pada minggu kedua demam</li></ul>- dipakai untuk mendeteksi karier<br />Pemeriksaan mikrobiologi<br />pendahuluan<br />15<br />15<br />
  21. 21. 4. Kultur sumsum tulang<br />- lebih sensitif 80-95%<br /><ul><li> tidak dipengaruhi: periode perjalanan penyakit</li></ul> saat spesimen diambil, pengobatan dengan<br /> antimikroba sebelumnya<br /><ul><li> memerlukan ketrampilan dan pengalaman</li></ul> khusus<br /><ul><li> invasif</li></ul>Pemeriksaan mikrobiologi<br />pendahuluan<br />16<br />16<br />
  22. 22. 5. Karakteristik koloni<br />17<br />
  23. 23. 6. Identifikasi biokimia<br />Ket: Alk=alkaline, Wk=lemah, V= hasil bervariasi<br />18<br />
  24. 24. Pemeriksaan Serologi<br />1. Identifikasi Serologi Salmonella<br />19<br />
  25. 25. 2. Felix-Widal<br />Melacak kenaikan titer antibodi<br />Sensitivitas 53 – 89 %, spesifisitas 89 – 98 %<br />20<br /><ul><li> Negatif palsu: pemberian antibiotika
  26. 26. Positif palsu: Salmonella serotipe lain yang memiliki antigen O dan H, reaksi silang epitop Enterobacteriacae lain</li></li></ul><li><ul><li> Deteksi antibodi dan antigen
  27. 27. Prinsip: Immunoassay Magnetic Binding Inhibition (IMBI)
  28. 28. Mengukur kemampuan serum antibodi IgM/ antigen 09 menghambat reaksi antara antigen berlabel lateks magnetik (reagen warna coklat) dan monoklonal antibodi berlabel lateks warna (reagen warna biru)
  29. 29. Tingkat inhibisi=konsentrasi antibodi Ig M/ antigen 09 Salmonella Typhidalam sampel
  30. 30. membandingkan warna akhir terhadap skala warna</li></ul>3. IDL Tubex<br />21<br />21<br />
  31. 31. Gambar 6. IlustrasiTUBEX TF mendeteksi antibodiO9 (Sumber Tam F 2008)<br />22<br />3. IDL Tubex<br />
  32. 32. 23<br />3. IDL Tubex<br />Gambar 8. SkorTUBEX TF dan respon terbentuknya antibodi<br />(Sumber : Lim 2010)<br />
  33. 33. sensitifitas dan spesifitas tinggi<br />deteksi antibodi (IgM dan IgG) terhadap S. Typhi:anti LPS, anti OMP dan anti flagelin<br />deteksi antigen: antigen O, OMP, dan flagelin<br />4. Uji enzyme-linked immunosorbent assay (ELISA)<br />24<br />
  34. 34. deteksi antibodi IgM dan IgG spesifik S. Typhi terhadap antigen 50 kD OMP<br />sederhana, cepat, murah, diagnosis dini<br /> sensitivitas 95%, spesifisitas 75%, nilai prediksi positif dan negatif tinggi<br />IgM (+)menunjukkan infeksi fase awal<br />IgG (+) dan IgM (+): infeksi akut pada fase pertengahan infeksi<br />daerah endemik: IgG meningkat, tidak dapat membedakan kasus akut dan konvalesen<br />5. Typhidot<br />25<br />
  35. 35. 26<br />goat anti-rabbit IgG<br />anti human IgG<br />anti-human IgM monoklonal<br />antigen H dan O S.Typhidiikatkan colloidal gold dan rabbit IgG- gold conjugates<br />6. Immunochromatographic Assay<br />
  36. 36. deteksi antibodi IgM spesifik S. Typhi pada serum atau darah utuh<br />7. Ig M dipstick<br />27<br />
  37. 37. Bakteriofag (faga): virus yang menginfeksi bakteri<br />Dapat menimbulkan lisis pada kuman S.Typhi yang mengandung antigen Vi<br />Dipakai faga Vi II<br />Stabil, pengerjaan sulit<br />Faga Indonesia: D2,A,B1,D6 dan E1<br />Isolat S.Typhi Indonesia jenis faga mirip negara di Asia Tenggara<br />Jenis Faga S.Typhi<br />28<br />
  38. 38. Pelacakan DNA S.Typhi<br />Polymerase Chain Reaction (PCR) <br />Replikasi DNA menggunakan primer spesifik<br />Sensitivitas dan spesifisitas tinggi<br />Memerlukan ketrampilan dan ketelitian tinggi<br />Lebih mahal<br />Gen pembawa faktor virulen mengelompok dalam suatu pathogenecity island (PI) <br />29<br />
  39. 39. Tes Kepekaan Antibiotika<br /> Penting <br /> terapi<br /><ul><li>Banyak multidrug-resistant (MDR)
  40. 40. Resisten: ampicillin, chloramphenicol, sulfonamide, trimethoprim, streptomycin dan tetracycline</li></ul>Obat alternatif:<br /><ul><li> fluoroquinolones
  41. 41. Cephalosporin generasi III
  42. 42. monobactam beta-lactam
  43. 43. macrolide </li></ul>30<br />
  44. 44. CLSI:<br />Antibiotika:berdasarkan obat yang biasa digunakan <br />Isolat feses: rutin dilaporkan ampicillin, quinolone, trimethroprim-sulfamethoxazole<br />Isolat ekstraintestinal: ditambah chloramfenicol, dan chepalosporine generasi ketiga <br />Tes Kepekaan Antibiotika<br />31<br />
  45. 45. terimakasih<br />32<br />
  46. 46. 33<br />
  47. 47. LPS<br />Gastrointestinal<br />epithelium<br />M cell<br />APC<br /> MHC class II<br />Ag<br />TCR<br />T Helper <br />/ CD4 T cell<br />TH1<br />TH2<br />TH3<br />IL-4, IL-6, IL-10<br />IL-2, TNF-, INF-<br />T cytotoxic<br />/ CD8 T Cell<br />B cell<br /> MHC class I<br />TGF-<br />IgM<br />Target cell<br />IgG<br />Cellular <br />immunity<br />Humoral <br />immunity<br />IgA<br />The concept of the specific immune response<br />Sumber: Judayana 2008<br />
  48. 48. sensitifitas dan spesifitas tinggi<br />indirect ELISA: mendeteksi antibodi (IgM dan IgG) terhadap S. Typhi<br />Antigen dilekatkan pada fase padat<br />Ditambah serum penderita yang berisi antibodi spesifik terhadap antigen S.Typhi<br />Ditambah konjugat anti human IgM atau IgG berlabel enzim<br />Pemberian substrat sesuai dengan enzim yang dilabel pada konjugat, menyebabkan enzim memecah substrat dan terjadi perubahan warna yang dibaca melalui fotometer<br />Uji enzyme-linked immunosorbent assay (ELISA)<br />35<br />
  49. 49. Ab II berlabel enzim<br />Ag<br />SUBSTRAT berkromogen<br />Ab I pada Fase padat<br />PRODUK berwarna<br />Prinsipdasar (taklangsung)<br />double antibody sandwich ELISA<br />
  50. 50. RAPID DIAGNOSIS OF TYPHOID FEVER BY ENZYME-LINKED IMMUNOSORBENTASSAY DETECTION OF SALMONELLA SEROTYPE TYPHI ANTIGENS IN URINE Am. J. Trop. Med. Hyg., 70(3), 2004, pp. 323–328<br />The specificity of the Vi assay exceeded 90% when assessedamong febrile and healthy control subjects without blood culture-confirmed typhoid fever. However, surprisingly, thespecificity was only 25% when assessed among patients withblood culture-confirmed brucellosis, despite the use of monoclonalantibodies to Vi (an improvement to the ELISA suggestedby other investigators).20 This indicates that Brucellaspecies may carry a Vi-like antigen, or that a Vi-like antigenis released into the urine of patients with bloodstream infections,perhaps as part of the renal component of the acutephase response. We were unable to find previous reports ofVi-like antigen from the urine of patients with brucellosis.<br />Explanations include that brucellosis patients have neverbeen selected as a control group when evaluating ELISAs forthe diagnosis of typhoid fever. Positive reactions persistedeven after experiments to address Brucella interference orcross- reactivity. This included absorption of the anti-Vi polyclonalantibody with attenuated Brucella cells which alsocompromised S. Typhi typhoid reactivity.<br />37<br />
  51. 51. 38<br />
  52. 52. 39<br />
  53. 53. Enteropathogenic E. coli, Salmonella, and Shigella: Masters of Host CellCytoskeletal Exploitation Emerging Infectious Diseases Vol. 5, No. 2, April.June 1999<br />Bacterial Factors Involved in Salmonella Invasion<br />Salmonella entry into nonphagocytic epithelialcells requires several chromosomal genes(inv/spa) clustered in a pathogenicity island termed SPI1 (Salmonella pathogenicity island 1) (34). Like EPEC, SPI1 encodes a type IIIsecretion system and several potential virulencefactors secreted by this machinery. The type IIIsecretionsystem is activated upon host-cellcontact and allows export of virulence determinantsdirectly into the host cell, where they effectbacterial uptake (35,36). Recently, SptP, abacterial protein encoded within SPI1, has beenshown to be translocated into the host epithelialcell, where it modulates the host actincytoskeleton through its tyrosine phosphataseactivity (37) (Figure 2B). Disruption of a criticalCys residue in the catalytic domain of SptPresults in loss of phosphatase activity (38). It ishypothesized that SptP may function indisrupting host actin stress fibers, therebyfacilitating membrane ruffling and subsequentbacterial uptake into host cells.<br />40<br />
  54. 54. Other bacterial factors are not encoded nextto the secretion apparatus but instead on thegenome of a cryptic bacteriophage found in the Salmonella chromosome. <br />Recently, a virulence factor encoded within this genome, SopE, hasbeen shown to be required for efficient bacterialentry into host cells (39). SopE requires the typeIII-secretion system to be translocated into thehost cell, where it can directly stimulate actincytoskeletal rearrangements. It acts as aguanidine exchange factor for members of theRho subfamily of small GTPases. sopE mutantsexhibit less extensive actincytoskeletal rearrangementsupon entry into epithelial cells thando wild-type Salmonella (40). <br />This discoveryclearly illustrates how pathogens (which containno primary sequence homology with hostproteins) can craftily subvert the host.s ownsignaling machinery within the cell by mimicking host proteins<br />41<br />
  55. 55. Host Factors Involved in Salmonella nvasion<br />The massive restructuring of the host cytoskeletal components during Salmonella entry requires many host factors. A Rhosubfamily member, Cdc42, is needed formediating bacterial uptake through membraneruffling (41). It is believed that the guanidineexchange activity of SopE is responsible for thestimulation of Cdc42 in the host. The pathogenalso activates host PLC upon bacterial contact,leading to the production of two secondmessengers, which further initiate signalingevents (42). As a consequence, the host cell.s Ca2+levels are altered to trigger cytoskeletal rearrangements resulting in Salmonella invasion.<br />Although EPEC and Salmonella use someof the same signaling components (PLC, Ca2+fluxes), the cytoskeletal changes induced in thehost cell by each pathogen are quite different.This could be the result of different upstream ordownstream effectors in the signaling pathway.Several cytoskeletal components involved ininvasion have been identified. These include a-actinin, tropomyosin, ezrin, andtalin (19). Thespecific roles of these proteins in Salmonella invasion are not defined.<br />42<br />
  56. 56. 43<br />
  57. 57. Antigenic formulas of the Salmonella serovars (Kauffmann-White scheme)<br />Nomenclature of the genus Salmonella<br />The genus Salmonella encompasses three species: <br />.Salmonellaenterica which is divided into six subspecies: (i) Salmonellaenterica subsp. arizonae, (ii) Salmonellaenterica subsp. diarizonae, (iii) Salmonellaenterica subsp. enterica, (iv) Salmonellaenterica subsp. houtenae, (v) Salmonellaenterica subsp. indica, and (vi) Salmonellaenterica subsp. salamae. <br />.Salmonellabongori. <br />.Salmonellasubterranea. <br />This nomenclature reflects present Salmonella taxonomy.<br />Nomenclature of serovars<br />The strains of Salmonella are serologically classified into serovars (serotypes) using the Kauffmann-White scheme which is defined and maintained by the WHO Collaborating Centre for Reference and Research on Salmonella at the Pasteur Institute, Paris, France. There are 2,541 serovars (serotypes) of Salmonella reported in 2002 and new serovars are listed in annual updates of the Kauffmann-White scheme. All serovars are expressed by an antigenic formula. Serovars of subspecies enterica are given a name, whereas the remaining serovars are designated by only their antigenic formula<br />44<br />
  58. 58. The subspecies of Salmonellaenterica are represented by Roman numerals in the scheme:      .S. enterica subsp. enterica = I      .S. enterica subsp. salamae = II      .S. enterica subsp. arizonae = IIIa      .S. enterica subsp. diarizonae = IIIb      .S. enterica subsp. houtenae = IV      .S. enterica subsp. indica = VI <br />Salmonellabongori is represented by Roman numeral V. <br />Some names appearing in old editions of Kauffmann-White scheme are now deleted :      . Serovars names given to variants converted by lysogenization. Ex. Newhaw is now called Muenster var. 15+, Arkansas is now called Muenster var. 15+,34+ as they correspond to variants od serovar Muenster converted by pahe ε15or by phages ε15 + ε34, respectively.      . Serovars names given before 1966 to serovars later found not to belong to subspecies Salmonellaenterica subsp. enterica. <br />Sqare brackets "[]" are used for O or H factors that may be present or absent with or without relation to phage conversion. When H factors are in square brackets, this means that they are exceptionally found in wild strains. <br />Brackets "()" are used for O and H factors weakly agglutinable. <br />Designation of the O groups<br />The first-described O group were designated by letters of the alphabet. Later it was necessary to continue with numbers 51 to 67. It is now more logical to call each O group using the characteristic O factor. Letters are usually kept into brackets, Ex. O:4 (B), O:21 (L). <br />45<br />
  59. 59. pulse-temperature dissociation<br />Background The pulse rate ↑ 15-20 beats/min. for each degree ↑ in core body temperature > 39ºC; a lower than normal ↑ in pulse rate or relative bradycardia–ie, a PTD is not uncommon and occurs in burns, drug fever, hepatitis, intoxication–eg, trinitrotoluene, TNT, Legionnaires' disease, malaria–blackwater fever, acute MI, psittacosis, typhoid fever, yellow fever; relative tachycardia is far less common, but is typical of clinically silent PE, diphtheria and clostridial infections<br />46<br />
  60. 60. IgM dipstick<br />The assay is performed by incubation of the wetted test strip in a mixture of serum anddetection reagent, the serum being diluted at 1:50 in the detection reagent. Whole bloodmay be tested at a 1:25 dilution in detection reagent. The incubation period is threehours at room temperature. When incubation is complete the test strip is rinsedthoroughly with water and then allowed to dry. The result is read by visual inspectionof the test strip for staining of the antigen and control lines. The test result is scorednegative if no staining of the antigen line occurs and is graded 1+, 2+, 3+ or 4+ if thereis weak, moderate strong or very strong staining as indicated by comparison with acoloured reference strip. The control line should stain in all runs.<br />47<br />
  61. 61. IgM dipstick<br />Evaluations of the dipstick test in laboratory-based studies in Indonesia (31, 32),Kenya (33), Viet Nam (33) and Egypt (34) have shown consistent results. These studiesindicated sensitivities of 65% to 77% for samples collected at the time of firstconsultation from culture-confirmed patients and specificities of 95% to 100%. Theresults of culture and serological investigation may be influenced by various factors,among them the time of sample collection and the use of antibiotics before consultationand sample collection. In a study conducted in Makassar, Indonesia, the sensitivity ofthe blood culture method was estimated to be 66%, and that of the dipstick testcalculated for the combined group of culture-confirmed and culture-negative patientswith a final clinical diagnosis of typhoid fever was 48%. The sensitivity ranged from29% for samples collected during the first week of illness to 96% for samples collectedat a later stage. Tests on follow-up samples showed seroconversion in the majority of the dipstick-negative typhoid patients. <br />48<br />
  62. 62. IgM dipstick<br />The dipstick test provides a rapid and simple alternative for the diagnosis of typhoidfever, particularly in situations where culture facilities are not available. The assay canbe performed by people without formal training and in the absence of specializedequipment. Electricity is not required, as the components can be stored without cooling.The results of the dipstick test can be obtained on the day when patients present.This makes prompt treatment possible. Specific antibodies usually only appear a weekafter the onset of symptoms and signs. This should kept in mind when a negativeserological test result is being interpreted.<br />49<br />
  63. 63. RAPID SEROLOGIC DIAGNOSIS OF PEDIATRIC TYPHOID FEVER IN AN ENDEMICAREA: A PROSPECTIVE COMPARATIVE EVALUATION OF TWO DOT-ENZYMEIMMUNOASSAYS AND THE WIDAL TESTAm. J. Trop. Med. Hyg., 61(4), 1999, pp. 654–657<br />The dot-EIA tests were performed using standard commercialkits (Typhidott and Typhidot-Mt (generous donations from Malaysian Biodiagnostics Research, Kuala Lumpur, Malaysia) containing nitrocellulose strips dotted with0.3 mg of the 50-kD OMP. <br />The Typhidottstrips were probedwith 1:100 dilution serum, washed with phosphate-bufferedsaline, and 1 hr later the antigen-antibody complexes werevisualized by addition of horseradish peroxidase–conjugatedantiserum to human IgG and IgM and 4-chloro-1-naphthol.Positive results were read in comparison with control seraand represented a titer . 1:100. A positive IgG or IgM result,either alone or in combination, was regarded as a positivetest result in our patients. <br />The Typhidot-Mt differed inthe initial steps: the addition of an IgG-inactivation complex and subsequent removal of the bound-IgG with anti-humanIgG labeled with horseradish peroxidase. The subsequent antibodybinding and color change identified primarily IgM antibodies against the OMP<br />50<br />
  64. 64. Antibiotic therapy has been shown to alter the antibody response to S. typhi infection, and given that a large number of the patients hadpreviously received antibiotics, this factor may have alteredthe antibody titers against O antigens<br />Our findings of sensitivity and specificity were also lower than values .<90%reported recently by Karamat and others from northernPakistan. These differences may be due to several factorsincluding the genomic diversity among S. typhi isolates inthe region28 and differences in antigenic epitopes. <br />Other factorsresponsible for reported differences in areas of high endemicityare various stages of the illness and the rate ofincrease of IgG antibodies to the OMPs, which may interferewith identification of concomitant IgM antibodies. Most ofour patients presented in the second week of their illness,whereas information on duration of illness is lacking in other studies<br />51<br />RAPID SEROLOGIC DIAGNOSIS OF PEDIATRIC TYPHOID FEVER IN AN ENDEMICAREA: A PROSPECTIVE COMPARATIVE EVALUATION OF TWO DOT-ENZYMEIMMUNOASSAYS AND THE WIDAL TESTAm. J. Trop. Med. Hyg., 61(4), 1999, pp. 654–657<br />
  65. 65. Salmonella Typhi IgMELISA<br />PRINCIPLES OF THE TEST<br />Diluted patient serum (serum diluent contains sorbent to remove rheumatoid factor and human IgGinterference) is added to wells coated with purified antigen. IgM specific antibody, if present, binds to theantigen. All unbound materials are washed away and the enzyme conjugate is added to bind to theantibody-antigen complex, if present. Excess enzyme conjugate is washed off and substrate is added. Theplate is incubated to allow the hydrolysis of the substrate by the enzyme. The intensity of the colorgenerated is proportional to the amount of IgM specific antibody in the sample<br />Antibody Index Interpretation<br /><0.9 No detectable antibody to Salmonella typhiIgM by ELISA.<br />0.9-1.1 Borderline positive. Follow-up testing is recommended if clinically indicated.<br />>1.1 Detectable antibody to Salmonella typhiIgM by ELISA.<br />LIMITATIONS OF THE TEST<br />1. To enhance sensitivity and specificity of this IgM test provided sample diluent has been formulated toblock IgG and Rheumatoid Factor (RF) interferences. Turbidity could be seen after diluting serum withsample diluent. This turbidity is due to the blocking of serum IgG and has shown no interference withtest results. It can be removed by centrifugation.<br />2. In specimens with high RF and high autoimmune antibodies, the possibility of eliminating theinterferences cannot be ruled out entirely.<br />3. The test results obtained using this kit serve only as an aid to diagnosis and should be interpreted inrelation to the patient’s history, physical findings and other diagnostic procedures.<br />4. Lipemic or hemolyzed samples may cause erroneous results.<br />52<br />
  66. 66. ELISA<br /> In-house indirect sandwich ELISAs were established to detect anti-LPS IgA, IgM, and IgG and antiflagellumIgG. <br />All reagents used were purchasedfrom Sigma unless stated otherwise. Immulon 1b flat-bottom 96-well microtiterplates (Dynex Technologies, Billinghurst, United Kingdom) were coated overnightat 4°C with 100 ml of either 1 mg of antigen/ml in coating buffer (0.1 Mcarbonate buffer [pH 9.4], antigen positive) or coating buffer alone (antigennegative). <br />The plates were blocked for 1 h at 37°C with 200 ml of phosphatebufferedsaline containing 1% bovine serum albumin (BSA). Sera were eitherassayed at a single dilution (1/1,000 for anti-LPS IgG, 1/500 for anti-LPS IgA, or1/250 for anti-LPS IgM and antiflagellumIgG) or serially diluted (starting at adilution of 1/50). <br />53<br />
  67. 67. Sera were diluted in phosphate-buffered saline containing 0.1%BSA and 0.05% Tween 20, 100 ml was applied to the appropriate wells, and theplates were incubated for 4 h at room temperature. Bound antibodies (IgA, IgG,or IgM) were detected using heavy-chain-specific goat antibodies directly conjugatedto alkaline phosphatase. The latter were diluted (anti-IgG, 1/5,000; anti-IgA, 1/500; and anti-IgM, 1/2,500) in Tris-buffered saline containing 0.1% BSAand 0.05% Tween 20. <br />One hundred microliters was added to each well, and theplates were incubated overnight at 4°C. One hundred microliters of p-nitrophenylphosphate (1 mg/ml) was added to each well, and the plates were incubatedat ambient temperature in the dark for 30 to 40 min. The absorbance at 405 nm(reference filter, 450 nm) was determined using an automated ELISA reader (Bio-Rad)<br />54<br />
  68. 68. For sera assayed at a single dilution, antibody levels were expressed in opticaldensity (OD) units. These were taken as the mean OD of three wells with antigenminus the OD of a single well without antigen. For the titration assays, sera wereassayed in triplicate (two wells antigen positive and one well antigen negative),and the titer was taken as the highest dilution giving a net OD (mean OD ofantigen-positive wells minus OD of antigen-negative well) of ≥ 0.3 (anti-LPSIgG) or ≥ 0.2 (all other antibodies). Six standards were included on each plate,and the OD or titer of the samples was adjusted accordingly. Blank wells with nosera were included to monitor background.<br />55<br />
  69. 69. Serotype Typhi IgM dipstick assay.<br />The serotype Typhi IgM dipstick assay was provided by H. L. Smits, Department of Biomedical Research, Royal TropicalInstitute, Amsterdam, The Netherlands. Briefly, serum samples were diluted(1/50) in the detecting reagent (containing dye-labeled, anti-human IgM antibodies).<br />Nitrocellulose dipsticks coated with heat-inactivated serotype Typhiwere immersed in the diluted serum and incubated at room temperature for 4 h.<br />The strips were then washed and dried at room temperature. The sera weregraded (0 to 4) according to the staining intensity of the colored band correspondingto the antigen. <br />The test was performed by a single operator, and theresults were interpreted by three members of the laboratory staff who had noknowledge of the samples being tested.<br />56<br />
  70. 70. Demam Tifoid dan lnfeksi Lain dari Bakteri SalmonellaKenneth C. Hinton, MD, FAAP<br />57<br />
  71. 71. Faktor Penentu Virulensi S. typhi<br />Somatik (O), ada 2 determinan antigen (endotoksin-LPS. Acetylated glucosamine disaccharide-lipid A)<br />Kapsular Vi<br />Polysaccharide (Vi) ada 2 determinan antigen (O-acetyl, <br />N-acetyl carboxyl)<br />Outer Membrane Proteins (OMPs)<br />Flagela protein (H). antigen (fase 1 & H)<br />Non - Porin<br />Porin <br />(OmpB,C,D,F, dan OmpR)<br />Sumber: Sarasornbatlv, 1992; Levinson, 2003; Ryan, 2003 denganmodifikasi).<br />58<br />
  72. 72. 59<br />
  73. 73. 60<br />Sensitivity of TUBEX TF vs Antibody respond & Days after infection<br />Sources : Prof PL Lim, University of Hongkong<br />
  74. 74. 61<br />
  75. 75. In the  macrophages, the Salmonella typhi is able to replicate and stay in  the areas of   the spleen, liver, and bone marrow( Diagle et al. 2001).  <br />The macrophages can loose the ability to kill the intracellular bacteria. Therefore T helper 1 cells with the Fas ligand can initiate the caspase pathway and kill the macrophage ( Janeway et al. 2002).  <br />The inflammation at the site of infection may be caused the production of  Interleukin 1 , Interleukin 18 by caspase 1, TNF alpha, and the release of the contents of bacteria from the macrophages ( Brennan et al. 2000).When the bacteria is able to reenter circulation and make its way back into the blood, one is then able to view the clinical onset of the disease such as fever, nausea, constipation and diarrhea. The Salmonella typhi  is then removed from the blood via the gall bladder to the small intestine.<br />62<br />
  76. 76. Penyebab ulcus demam tifoid<br />When the bacteria reaches the Peyers patches a second time, The macrophages/ T helper  and Cytotoxic CD8+ T cell accumulate and can try and kill the Salmonella typhi. The localization to the small intestine leads to Salmonella typhi being exposed a second time to Peyers patches causing  inflammation, ulceration, and  typhoid ulcers. The inflammation is once again caused by the release of cytokines such as Interferon gamma and tumor necrosis factor alpha , which lead to vascularization, the accumulation of more macrophages, and eventually necrosis ( Shtrichman et al. 2002). <br />The ulcers can hemorrhage and this usually occurs during this time. The (Macdonald et al. 2000). ulcerations are caused by the production of the enzyme, matrix metalloproteinases and this caused the loss of the mucuosal membranes in the small intestineThe loss of the mucosal membranes lead to ulcerations.<br />The Peyers patches perforate causing generalized peritonitis and septicaemia, the commonest cause of death in typhoid fever (Butler et al. 1991).The death occurs  in <5% of patients, but with a mortality approaching 40% and increasing substantially (83%) if treatment is delayed longer than 96 hours ( Van Basten et al. 1994).<br />63<br />
  77. 77. Case definition (WHO 2003)<br />Confirmed case of typhoid fever<br />A patient with fever (38°C and above) that has lasted for at least three days, with a laboratory-confirmed positive culture (blood, bone marrow, bowel fluid) of S. typhi.<br />Probable case of typhoid fever<br />A patient with fever (38°C and above) that has lasted for at least three days, with apositive serodiagnosis or antigen detection test but without S. typhi isolation.<br />Chronic carrier<br />Excretion of S. typhi in stools or urine (or repeated positive bile or duodenal stringcultures) for longer than one year after the onset of acute typhoid fever. Short-termcarriers also exist but their epidemiological role is not as important as that of chroniccarriers. Some patients excreting S. typhi have no history of typhoid fever.<br />64<br />
  78. 78. Diagnosis of typhoid carriers<br />Carriers of S. typhi are either convalescent carrierswho excrete the organism for a limited period oftime after apparent clinical cure, or chronic carriersin whom persistent excretion of S. typhi in stool orurine can be detected a year after clinical illness.<br />Chronic faecal carriers occur more commonly thando chronic urinary ones. The numbers of typhoidbacilli excreted in the stools of these cases maybe inordinately large, each gram of faecesusuallycontaining 10 or more viable organisms.<br />Thediagnosis of carrier status is established byculturing the organism from the relevant specimen of the suspected person.<br />65<br />
  79. 79. Gelatin capsule string test is preferable fordetection of chronic faecal carrier. Becauseexcretion of organisms in the faeces of chroniccarriers is often intermittent, methods other thanfaecalcultures have been devised to increase thesensitivity of culture. <br />One such method is to culturethe duodenal aspirates in suspected gall bladdercarriers. Gilman et al12 have suggested the use ofa gelatin capsule containing a nylon string forcollecting duodenal specimens. This technique hasbeen found to be highly sensitive and also it canbe used for giardiatrophozoitedemonstration simultaneously.<br />66<br />
  80. 80. Vi-antibody tests<br />Serological tests are used to screen peoplesuspected of being chronic carriers of Salmonellatyphi. The Vi agglutination test has been used formany years. The Vi test should not be usedindiscriminately in screening populations fortyphoid carriers, but may have limited usefulnessin an attempt to trace suspected carriers.<br />A Vireactor must be followed-up by bacteriologicalinvestigations, but the chance of reactors being acarrier is very small. The demonstration of a carrieramong the reactors does not exclude the possibilityof another carrier occurring among the nonreactors.The sensitivity of Vi antibody detection isnot more than 70%. <br />67<br />
  81. 81. Moreover the test is not 100%specific too as it has been found false positive infew culture negative cases. <br />There are various testsdeveloped to detect this antigen which include passive haemagglutination, solid phase radioimmunoassay, counter immunoelectrophoresis, and recently the ELISA.<br />All these testshave variable sensitivity and specificity. Vi antigenof Citrobacter coated on red blood cells has alsobeen used to demonstrate the anti-Vi antibodies in blood3,12.<br />68<br />
  82. 82. 69<br />
  83. 83. 70<br />
  84. 84. 71<br />
  85. 85. Uji Diazo urine<br />Pemeriksaan yang relatifmurah, mudahdansederhana, sehinggadapatdikembangkanpemakaiannyaditempatdenganfasilitaslaboratoriumsederhana.<br />Reaksidiazopositifpada urine penderitademamtifoiddidugadisebabkanolehadanyasuatuzat yang mengandungcincin phenol. Zatiniberasaldarihasilpembusukan protein dalamusus yang diserapkedalamalirandarahkemudianmengalamidetoksifikasidihatidandikeluarkanmelaluiginjal. Ujidiazobiasanyamemberikanhasilpositifpadaharike 5 - 14 perjalananpenyakitdemamtifoiddanmenjadinegatifbilademammenghilang. <br />Beberapapenyakit yang seringmemberikanhasilpositifpalsupadaujidiazoadalahmorbilli, tuberkulosismiliarakutkadangjugademamberdarah dengue. Padapenderitademamtifoid yang mengalamirelaps, ujidiazoakankembalimenjadipositif. Obatgolonganantipiretika, antibiotikagolongan sulfa jugamemberikanhasilpositifpalsu. Untukmendapatkanhasil yang baikharusdigunakanreagendiazo yang baru. <br />72<br />Sumber: Muljati M 2010<br />
  86. 86. Correlation of Clinical and Hematologic Profiles with Bone Marrow Responses in Typhoid FeverJoseph James, Tarun Kumar Dutta AND S. Jayanthi <br />peripheral blood cytopenias that were not accompanied byconcurrent bone marrow suppression, suggesting a peripheralmechanism responsible for the blood dyscrasia in those cases.<br />No patient had evidence of disseminated intravascular coagulopathy.<br />There was no correlation between clinical severity of diseaseand bone marrow changes. Bone marrow/peripheral blood changesdid not influence the outcome of the disease, since all thepatients recovered fully after treatment<br />73<br />
  87. 87. Rose spots<br />Rose spots are red macular lesions 2-4 millimeters in diameter occurring in patients suffering from enteric fever (which includes typhoid and paratyphoid). These fevers occur following infection by Salmonella typhi and Salmonella paratyphi respectively. Rose spots may also occur following invasive non-typhoid salmonellosis.<br />Rose spots are bacterial emboli to the skin and occur in approximately 1/3 of cases of typhoid fever. They are one of the classic signs of untreated disease, but can also be seen in other illnesses as well including shigellosis and nontyphoidal salmonellosis. They appear as a rash between the seventh and twelfth day from the onset of symptoms. They occur in groups of five to ten lesions on the lower chest and upper abdomen, and they are more numerous following paratyphoid infection. Rose spots typically last three to four days.<br />74<br />
  88. 88. UJI SEROLOGI DEMAM TIFOID<br />Dr. JusakNugraha, dr, MS,SpPK (KGH,KIm,KT-I)<br />Bagian Patologi Klinik<br />FK UNAIR / RSUD Dr. Soetomo, Surabaya<br />75<br />
  89. 89. PEMERIKSAAN LABORATORIUM DEMAM TIFOID<br />TES WIDAL<br />TES IgM Salmonella<br />ELISA<br />Deteksicepat Antigen (reversed serology)<br />BIAKAN / KULTUR DARAH<br />PCR<br />BIAKAN URINE / TINJA<br />Dr. JusakNugraha, dr, MS,SpPK (KGH,KIm,KT-I)<br />76<br />
  90. 90. INDIKASI<br />MembantumenegakkanDx & menentukanPxdaripenyakit DEMAM TIFOID<br />SUPAYA MENDAPAT PENGERTIAN YANG JELAS MENGENAI LANDASAN DASAR& INTERPRETASI TES, PERLU DIKETAHUI :<br />- Sifat-sifatantigenikdariS. typhi/paratyphi<br />- PATOGENESIS demamtifoid<br />Dr. JusakNugraha, dr, MS,SpPK (KGH,KIm,KT-I)<br />77<br />
  91. 91. Dr. JusakNugraha, dr, MS,SpPK (KGH,KIm,KT-I)<br />Gambar 34. Struktur antigen Salmonella<br />78<br />
  92. 92. Dr. JusakNugraha, dr, MS,SpPK (KGH,KIm,KT-I)<br />Gambar 35. Strukturskematikdarienvelopedandindingsel Salmonella Typhi<br />79<br />
  93. 93. Jenis Antigen<br />Antigen O" :: polysaccharides associated with the lipopolysaccharide (LPS) of the bacterial outer membrane. <br /> "H" antigens are proteins associated with the bacterial flagella (singular; flagellum).<br />Pathogenic strains of Salmonella typhi carry an additional antigen, "Vi", so-called because of the enhanced virulence of strains that produce this antigen, which is associated with a bacterial capsule.<br />80<br />
  94. 94. 81<br />Dr. JusakNugraha, dr, MS,SpPK (KGH,KIm,KT-I)<br />
  95. 95. Dr. JusakNugraha, dr, MS,SpPK (KGH,KIm,KT-I)<br />Tabel 1. Antigen Salmonella yang menyebabkan demam tifoid dan paratifoid<br />Tabel 2. Antigen O yang spesifik pada demam tifoid<br />(WHO, 2003. Background document : The diagnosis, treatment and prevention of typhoid fever)<br />82<br />
  96. 96. PATOGENESIS DEMAM TIFOID<br />Dr. JusakNugraha, dr, MS,SpPK (KGH,KIm,KT-I)<br />S. typhi 105<br />Sirkulasi Bakteriemi 1<br />Hepar<br />Lien<br />Organ2<br />d. thorac<br />Ren<br />Sutul<br />7-10 hari<br />Bakteriemi 2<br />Pl. Player<br />Gambar 36. Patogenesis demam tifoid<br />83<br />
  97. 97. BAKTERIEMI 2<br />KAPSEL Vi<br />Fagositosis sukar<br />Jalur alternatif komplemen<br />7 – 10 HARI ( PERUBAHAN KAPSEL )<br />Lisis<br />Fagositosis<br />Pembengkakan organ-organ<br />ENDOTOKSIN ( AG – O )<br />Sel-sel imunokompeten<br />Tak ada bakteriemi (akhir mgg. Ke 2)<br />AGLUTININ<br />O<br />H<br />Vi<br />GEJALA KLINIS<br />Gambar 37. Imunopatogenesisdemamtifoid<br />84<br />
  98. 98. Dr. JusakNugraha, dr, MS,SpPK (KGH,KIm,KT-I)<br />Gambar 38. Roseola di dada anak dengan demam tifoid<br />85<br />
  99. 99. Sensitivity of TUBEX TF vs Antibody respond & Days after infection<br />86<br />
  100. 100. PRINSIP DASAR UJI WIDAL yi. UJI AGLUTINASI<br />Dr. JusakNugraha, dr, MS,SpPK (KGH,KIm,KT-I)<br />Ag. pada permukaan sel<br />Aglutinasi<br />Ab.<br />Tak larut<br />Gambar 39. Prinsip dasar reaksi aglutinasi<br />87<br />
  101. 101. ANTIGEN UNTUK TES WIDAL :<br />1. Ag. O ( somatik ); S. typhi tak motil  + dg alkohol abs.<br />2. Ag. H ( flagella ); S. typhi yang motil  + dg formalin 0,3%<br />Pengawet larutan phenol 0,5%<br />3. Ag. PA; S.paratyphi A + dg formalin 0,3%<br />4. Ag. PB; S.paratyphi B + dg formalin 0,3%<br />Ad 1, sebelum dipakai  encerkan  kons. Alk, 12%<br />Dr. JusakNugraha, dr, MS,SpPK (KGH,KIm,KT-I)<br />88<br />
  102. 102. PROSEDUR PEMERIKSAAN<br />- DI ATAS GELAS OBYEK<br />- 2 TETES (  40 µl ) SERUM YG DIENCERKAN<br />- 2 TETES (  40 µl ) SUSPENSI ANTIGEN (O, H, PA, PB)<br />t° RUANGAN 5 MENIT<br />DIGOYANG<br />AGLUTINASI<br />Dr. JusakNugraha, dr, MS,SpPK (KGH,KIm,KT-I)<br />89<br />
  103. 103. Pengenceran serum:<br />O<br />Dimulai titer : 1: 40<br />H<br />Untuk :<br />Dimulai titer : 1: 80<br />PB<br />PA<br />Dimulai titer : 1:20<br />POSITIF<br />LANJUTKAN dg. PENGENCERAN SELANJUTNYA<br />DILAPORKAN : NEGATIF<br />NEGATIF<br />STOP<br />Dr. JusakNugraha, dr, MS,SpPK (KGH,KIm,KT-I)<br />90<br />
  104. 104. AMBANG ATAS NILAI NORMAL<br />- DILAKUKAN PADA 120 ORANG SEHAT<br />- AGLUTININ<br />O 1: 80<br />H 1: 80<br />PA 1: 40<br />PB 1: 160<br />Dr. JusakNugraha, dr, MS,SpPK (KGH,KIm,KT-I)<br />91<br />
  105. 105. KRITERIA DIAGNOSTIK DEMAM TIFOID<br />I. TITER AGLUTININ O atau<br /> 2Xambangatasnilai normal<br />AGLUTININ O&H <br />II. DALAM JANGKA WAKTU 5 - 7 HARI :<br />TITER MENINGKAT 4x <br />Dr. JusakNugraha, dr, MS,SpPK (KGH,KIm,KT-I)<br />92<br />
  106. 106. 2. Uji tabung.<br />: Pengenceran serial dalam 4 baris<br />Baris 1 diberi Ag O,<br />Baris 3 diberi PA,<br />Baris 2 diberi Ag H,<br />Baris 4 diberi PB,<br />2.1. CARA KLASIK :<br />Inkubasi ; 37°C, 24 jam<br />2.2 CARA STOKES :<br />Prinsip hampir sama dengan cara klasik<br />Dr. JusakNugraha, dr, MS,SpPK (KGH,KIm,KT-I)<br />93<br />
  107. 107. PERBEDAANNYA<br />a. Serumnya ; waterbath 56°C, ½ jam <br />b. Diluent-nya ; PBS, pH 7,4.<br />c. Inkubasinya ; tbg O  waterbath 37°C, 4 jam <br /> H, PA, & PB  waterbath 50°C, 2 jam<br />Pembacaannya : - Segera<br /> - Lebih baik setelah semalam pada 4°C<br />94<br />
  108. 108. `<br />Dr. JusakNugraha, dr, MS,SpPK (KGH,KIm,KT-I)<br />Gambar 41. Aglutinat di dasar tabung uji Widal yang positif<br />95<br />
  109. 109. Dr. JusakNugraha, dr, MS,SpPK (KGH,KIm,KT-I)<br />Gambar 42. Endapan antigen padadasartabungujiWidal yang positif<br />96<br />
  110. 110. FAKTOR-FAKTOR YANG PERLU DIPERHATIKAN.<br />4.1. Dr hasil 1X tes belum dapat ditarik kesimpulan yang berarti. Perlu ulangan setelah 5-7 hari<br />4.2. Harga normal tes Widal tabung.<br />Aglutinin O : 1/160<br />Aglutinin PA : 1/80<br />Aglutinin H : 1/160<br />Aglutinin PB : 1/320<br />4.3. Vaksinasi; aglutinin H dapat dipertahankan beberapa tahun,<br />4.4. Antibiotika ; dapat memperlambat kenaikan titer<br />Dr. JusakNugraha, dr, MS,SpPK (KGH,KIm,KT-I)<br />97<br />
  111. 111. 4.5. Febris ; dapat memberikan kenaikan titer<br />4.6. Narkotik ; Ketagihan  hasil pos. semu<br />4.7. Stadium penyakit ; mgg ke 1  neg./dlm batas normal <br /> mgg ke 2 titer mulai naik  puncaknya mgg ke 5 - 6;<br />4.8. Obatnya-2 imunosupresif; hambat pembentukan aglutinasi<br />4.9. Peny.-2 tertentu ; agamaglobulinemia & keganasan.<br />4.10. Infeksicampuran ; mis TB milliair sering neg. semu.<br />Dr. JusakNugraha, dr, MS,SpPK (KGH,KIm,KT-I)<br />98<br />
  112. 112. 5. KEPRAKTISAN<br />5.1. Kompleksitas pelaksanaan tes ; cukup sederhana<br />5.2. Waktu pemeriksaan ; relatif tidak lama<br />5.3. Peralatan ; tidak rumit.<br />5.4. Otomatisasi ; dapat semiotomatis dg lempeng mikrotitrasi & alat khusus<br />5.5. Tenaga terlatih ; sedang<br />5.6. Stabilitas reagensia ; cukup stabil<br />5.7. Perundang-undangan ; tak perlu peraturan tertentu.<br />Dr. JusakNugraha, dr, MS,SpPK (KGH,KIm,KT-I)<br />99<br />
  113. 113. IgM Salmonella<br /><ul><li>pemeriksaanantiboditerhadap antigen O9, lebihspesifikdariWidal
  114. 114. semi kuantitatif,</li></ul>Adabeberapametode:<br />Inhibitions Magnetic Binding Immunoassay (IMBI)<br />Dipstick assay (Immunochromatography / carikcelup)<br />Dr. JusakNugraha, dr, MS,SpPK (KGH,KIm,KT-I)<br />100<br />
  115. 115. Hasil: dalamskalawarna<br />Penetesan Brown Reagen 45 µl ke dalam V-shape wells.<br />Penambahan spesimen & kontrol 45 µl ke dalam V-shape dan dilarutkan.<br />Penetesan Blue Reagen 90 µl.<br />Inkubasi dan homogenasi 2 menit pada suhu kamar. <br />Pemisahan magnetik 5 menit dan pembacaan hasil. <br />tc<br />101<br />
  116. 116. Interpretasihasil<br />Pemisahan Magnetik<br />0<br />2<br />4<br />6<br />8<br />10<br />5 min<br />Interpretasi hasil<br />1<br />2<br />3<br />TUBEX<br />TF<br />4<br />8<br />0<br />Kontrol Neg<br />Kontrol Pos<br />Serum Sampel Pos<br />Skor 0-2: negatif, 3-10: positif<br />102<br />
  117. 117. Gambar 1a. Ilustrasiikataninhibisi antigen–antibodipadapemeriksaanantibodidan antigen<br />103<br />3. IDL Tubex<br />

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