Microbiological and Molecular Identification of Salmonella enterica serovar Typhi in Clinically Suspected Typhoid Patients in Garmiyan district A Thesis Submitted To The Council of the College of Science University of Sulaimani In Partial Fulfilment of the Requirements for the Degree of Master of Science (M.Sc.) in Biology/ Microbiology
By Hanan Tariq Subhei B.Sc. in Biology- 2003
Supervised by
Assist. Professor Dr. Bahrouz M. A. Jaff
2010 A.D.
1431 A.H.
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Salmonella enterica serovar Typhi
2003
2710
2010
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Salmonella enterica serovar Typhi
٢٠٠٣
1431
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2010
٦٠
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Dedication To my Father and mother, To My uncle Adnan To My brothers & sisters
Hanan
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Acknowledgements I would like to thank my advisor Dr. Bahrouz M.A.Jaff for his valuable guidance throughout my research. I would also like to thank Mr. Ahmed AL-Barzengi, Miss.Mustafa,S.. and Miss.Kareem, H. the teaching staff of both Biology Dept.,College of Education at Kalar, University of Sulimani, and Biology Department, College of science, University of Sulimani for their assistance. I wish to thank Dr.Abdulsalam A. from college of Agriculture, University of Sulimani and Miss. Layla the teaching staff of Kalar College of Education, University of Sulimani for their help in statistical analyses. My special thanks to laboratory staff in both Kalar General Hospital and Kalar district Commission\ Statistics directory for their assistance in this study. I would also like to express my sincere gratitude to all my friends.
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Summary The incidence rate of typhoid fever in Garmiyan district was 0.8, 0.57 and 0.52 per 105 for 2005, 2006 and 2007 respectively; it was higher in rural areas ( districts administratively belong to Kalar city) while for paratyphoid fever it was zero, 0.003 and 0.007 for the same years respectively. Summer season (June and July) showed high incidence of typhoid fever. Mortality rate of typhoid fever was zero, zero and 0.45 for the period 2005, 2006 and 2007. Paratyphoid fever appeared to be non fatal in the region. Out of 120 clinically suspected typhoid fever patients tested in the period September 2008 to May 2009, 70 (58.3%) were with significant titers for anti O and anti H antibodies (1:160) by Widal test. By using blood culture, biochemical tests and Remel RapID One system, Salmonella enterica serovar Typhi was isolated from 14 patients (11.6%) and primarily identified, Escherichia coli from 11 (9.16%) and Brucella from 7 (5.8%) patients. Six of the (14) Salmonella isolates were identified further by DNA amplification by PCR using the universal genus-specific primers (ConOriF/ ConOriR) for amplification of OriC. When the universal primers US/DS for amplification of hilA encode SPI-1 pathogenicity protein were used, four appeared positive. By using the variant Salmonella enterica serovar Typhi specific primers ST1/ST2 to amplify the gene Hd-1 that encodes the protein flagellin five isolates were positive. The same five isolates showed amplification also by using nested PCR to amplify the ST1/ST2 PCR product by ST3/ST4 primers. All the five isolates were positive for nested PCR were also showed amplification by using the primers tyv-s/tyv-as to amplify the gene which encodes O9 group. The only one isolate negative for variant specific primers revealed that more primers should be designed to confirm the
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isolate at the species level because it was identified as Salmonella enterica serovar Typhi by all microbiological methods used.
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2007 2006 2005 0,52 0,57 0,8 0,007 0,003 120 0,45 2009 2008 HO1:16058,370 Salmonella enterica 11,6 14 serovar Typhi Escherichia coli Remel RapID One 5,8 7
Brucella (9.16%) 11
14 PCRSalmonella
OriC
ConOriF/ ConOriR SPI- hilA US/DS Hd-1 ST1/ST2 1 Salmonella enterica serovar Typhi
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ST3/ST4 nested PCR ST1/ST2 O9tyv-s/tyv-as PCR Salmonella enterica serovar Typhi
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اﻟﺨﻼﺻﺔ ﻟﻘﺪ ﺟﺮى ﺣﺴﺎب ﺣﺪوث اﻟﺤﻤﻰ اﻟﺘﻮﻓﺎﻧﯿﺔ ﻓﻲ ﻣﻨﻄﻘﺔ ﻛﺮﻣﯿﺎن ﻣﻦ اﻗﻠﯿﻢ ﻛﺮدﺳﺘﺎن اﻟﻌﺮاق ،اذ ﻛﺎن ﺑﻤﻌﺪل 0,8و 0,57و 0,52ﻟﻼﻋﻮام 2005و 2006و 2007ﺑﺎﻟﺘﻌﺎﻗﺐ ،وﻛﺎن ﻋﺎﻟﯿﺎ ﻓﻲ اﻟﻤﻨﺎطﻖ اﻟﺮﯾﻔﯿﺔ ﻓﯿﻤﺎ ﻛﺎﻧﺖ ﻣﻌﺪﻻت اﻟﺤﻤﻰ اﻟﺸﺒﯿﮭﺔ ﺑﺎﻟﺘﻮﻓﺎﻧﯿﺔ ﻟﻼﻋﻮام ذاﺗﮭﺎ ﺻﻔﺮ و 0,003و 0,007ﺐﻗﺎﻌﺘﻟﺎﺑ .اظﮭﺮ ﺷﮭﺮا اﻟﺼﯿﻒ )ﺣﺰﯾﺮان و ﺗﻤﻮز( اﻋﻠﻰ ﻣﻌﺪل ﻟﺤﺪوث اﻟﺤﻤﻰ اﻟﺘﻮﻓﺎﻧﯿﺔ ﻓﯿﻤﺎ ﻛﺎن ﻣﻌﺪل اﻟﻮﻓﯿﺎت ﻟﮭﺬا اﻟﻤﺮض ﻟﻼﻋﻮام اﻟﺜﻼﺛﺔ ﺻﻔﺮ و ﺻﻔﺮ و 0,45ﺑﺎﻟﺘﻌﺎﻗﺐ ،وﺑﺪت اﻟﺤﻤﻰ اﻟﺸﺒﯿﮭﺔ ﺑﺎﻟﺘﻮﻓﺎﻧﯿﺔ ﻓﻲ اﻟﻤﻨﻄﻘﺔ ﻏﯿﺮ ﻣﮭﻠﻜﺔ .ﺑﯿﻦ 120ﺣﺎﻟﺔ ﻣﺮض ﻣﺸﺨﺼﺔ ﺳﺮﯾﺮﯾﺎ ﻟﻠﺤﻤﻰ اﻟﺘﻮﻓﺎﻧﯿﺔ ﻟﻠﻔﺘﺮة ﻣﺎﺑﯿﻦ ﺷﮭﺮي اﯾﻠﻮل 2008و ﻣﺎﯾﺲ ،2009ﻛﺎن (%58,3)70ﻣﻨﮭﻢ ذوي ﻣﻌﺎﯾﯿﺮ ﻟﻤﺴﺘﻀﺪات ) (Oو ) (Hﻣﺆﻛﺪة ﻟﻠﻤﺮض ) (1:160ﺑﺎﺳﺘﺨﺪام اﺧﺘﺒﺎر وﯾﺪال .و ﺑﺎﺳﺘﺨﺪام ﻣﺰرﻋﺔ اﻟﺪم و اﻻﺧﺘﺒﺎرات اﻟﻜﯿﻤﻮﺣﯿﻮﯾﺔ و ﻧﻈﺎم اﻟﺘﺸﺨﯿﺺ ، Remel RapID Oneﺟﺮى اﻟﻌﺰل و اﻟﺘﺸﺨﯿﺺ اﻻوﻟﻲ ﻟﺒﻜﺘﺮﯾﺎ Salmonella enterica serovar Typhi
ﻦﻣ 14ﻣﺮﯾﻀﺎ
) ،(%11,6ﻓﯿﻤﺎ ﻋﺰﻟﺖ ﺑﻜﺘﺮﯾﺎ Escherichia coliﻦﻣ (9.16%) 11و ﺑﻜﺘﺮﯾﺎ Brucella ﻦﻣ (%5,8) 7ﻰﺿﺮﻣ .ﻟﻘﺪ ﺗﺄﻛﺪ ﺗﺸﺨﯿﺺ ﺳﺘﺔ ﻋﺰﻻت ﻣﻦ ﺗﻠﻚ اﻻرﺑﻌﺔ ﻋﺸﺮ ﻟﺒﻜﺘﺮﯾﺎ Salmonellaﺑﺎﺳﺘﺨﺪام طﺮﯾﻘﺔ ﺳﻠﺴﻠﺔ ﺗﻔﺎﻋﻼت اﻟﺒﻠﻤﺮة ) (PCRﻟﺘﻜﺜﯿﺮﺟﯿﻦ OriCﺑﺎﺳﺘﺨﺪام اﻟﺒﺎدﺋﺎت ) (ConOriF/ ConOriRاﻟﻌﺎﻣﺔ اﻟﻜﺎﺷﻔﺔ ﻟﻠﺒﻜﺘﺮﯾﺎ ﻋﻨﺪ ﻣﺴﺘﻮى اﻟﺠﻨﺲ .و ﻋﻨﺪﻣﺎ اﺳﺘﺨﺪﻣﺖ اﻟﺒﺎدﺋﺎت ) (US/DSﻟﺘﻜﺜﯿﺮ اﻟﺠﯿﻦ hilAاﻟﻤﺸﻔﺮ ﻟﺒﺮوﺗﯿﻦ اﻻﻣﺮاﺿﯿﺔ SPI-1اظﮭﺮت ارﺑﻌﺔ ﻋﺰﻻت ﻧﺘﯿﺠﺔ اﯾﺠﺎﺑﯿﺔ .و ﺑﺎﺳﺘﺨﺪام اﻟﺒﺎدﺋﺎت ) (ST1/ST2اﻟﻤﺸﺨﺼﺔ ﻟﻠﺘﻐﺎﯾﺮ ) (Salmonella enterica serovar Typhiو اﻟﻤﻜﺜﺮة ﻟﺠﯿﻦ Hd-1اﻟﻤﺸﻔﺮ ﻟﺒﺮوﺗﯿﻦ اﻟﻔﻼﺟﻠﯿﻦ اظﮭﺮت ﺧﻤﺲ ﻋﺰﻻت ﻧﺘﯿﺠﺔ ﻣﻮﺟﺒﺔ ،وﻋﻨﺪﻣﺎ اﺟﺮﯾﺖ طﺮﯾﻘﺔ اﻟﺘﻜﺜﯿﺮ اﻟﻀﻤﻨﻲ )(nested PCR ﺑﺎﺳﺘﺨﺪام اﻟﺒﺎدﺋﺎت ) (ST3/ST4ﻟﻤﻨﺘﺠﺎت اﻟﺒﺎدﺋﺎت ) (ST1/ST2اظﮭﺮت اﻟﻌﺰﻻت اﻟﺨﻤﺴﺔ ﻧﺘﯿﺠﺔ ﻣﻮﺟﺒﺔ اﯾﻀﺎ .و اظﮭﺮت ﻛﺬﻟﻚ اﻟﻌﺰﻻت اﻟﺨﻤﺴﺔ ﻧﺘﯿﺠﺔ ﻣﻮﺟﺒﺔ اﯾﻀﺎ ﻟﻠﺘﻜﺜﯿﺮ ﺑﺎﺳﺘﺨﺪام اﻟﺒﺎدﺋﺎت ) (tyv-s/tyv-asاﻟﻤﺸﻔﺮة ﻟﻤﺴﺘﻀﺪ ) .(O9ان اﻟﻌﺰﻟﺔ اﻟﻮﺣﯿﺪة ﻣﻦ ﺑﯿﻦ اﻟﻌﺰﻻت اﻟﺴﺘﺔ و اﻟﺘﻲ ﻟﻢ ﯾﺠﺮي ﺗﺸﺨﯿﺼﮭﺎ ﺑﺎﺳﺘﺨﺪام اﻻﺧﺘﺒﺎرات اﻟﺘﺄﻛﯿﺪﯾﺔ ) (PCRو اﻟﺘﻲ ﻛﺎﻧﺖ ﻗﺪ ﺷﺨﺼﺖ ﻋﻠﻰ اﻧﮭﺎ ) (Salmonella enterica serovar Typhiﺑﺎﻟﻄﺮاﺋﻖ اﻟﺒﻜﺘﺮﯾﻮﻟﻮﺟﯿﺔ ﺗﺜﺒﺖ اﻟﺤﺎﺟﺔ ﻟﺘﺼﻤﯿﻢ اﻟﻤﺰﯾﺪ ﻣﻦ اﻟﺒﺎدﺋﺎت ﻣﻦ اﺟﻞ اﻟﺘﺸﺨﯿﺺ اﻟﺪﻗﯿﻖ ﻋﻨﺪ ﻣﺴﺘﻮى اﻟﻨﻮع.
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LIST OF CONTENTS Title
Page No.
Acknowledgments
I
Summary
II
List of contents
IV
List of tables
VIII
List of figures
IX
CHAPTER ONE: INTRODUCTION
1-2
CHAPTER TWO:LITERATURE REVIEW
3-22
2.1 A historical background 2.2 Typhoid fever 2.2.1 Pathogenicity 2.2.3 Epidemiology 2.2.3.1 Carriers 2.3 Salmonella enterica serovar Typhi 2.3.1 Taxonomy 2.3.2 Antigenic structure 2.3.2.1 Somatic(O) antigens 2.3.2.2 Surface (Envelope) antigens 2.3.2.3 Flagellar (H) antigens 2.4 Identification methods 2.4.1 Serological methods 2.4.1.1 Widal test 2.4.1.2 Enzyme-linked immunosorbent assay (ELISA) 2.4.1.3 Latex agglutination test 2.4.2Culture 2.4.2.1 Blood cultures 2.4.2.2 Stool cultures 2.4.2.3 Bone marrow cultures 2.4.2.4 Urine and other cultures 2.4.3 Molecular identification
3 4 4 6 7 7 8 10 10 11 11 13 13 14 14 15 16 16 17 17 17 18
2.4.3.1 Identification by Polymerase Chain Reaction (PCR)
18
2.5 Treatment
19
2.6 Vaccinations
21
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CHAPTER THREE: MATERIALS AND METHODS
23-43
3.1Materials
23
3.1.1Appartus and equipments
23
3.1.2Chemicals
24
3.1.3Culture media
25
3.1.4 Kits and enzymes
25
3.1.5 Primers
26
3.1.6 Widal test kit
26
3.1.7 Culture media
26
3.1.7.1 Brain heart infusion broth
26
3.1.7.2. Brain heart infusion agar
27
3.1.7.3 Biphasic blood culture
27
3.1.7.4 Blood agar
27
3.1.7.5 MacConkey′s agar
27
3.1.7.6 Salmonella Shigella agar
27
3.1.7.7 Kligler iron agar
27
3.1.7.8 Simmon′s Citrate agar
28
3.1.7.9 Indol Broth
28
3.1.7.10 Semi solid agar for motility test
28
3.1.7.11 Methyl Red-Voges Proskauer broth
28
3.1.7.12 Nutrient broth
28
3.1.7.13 Urea agar base
28
3.1.7.14 LB media
29
3.1.8 Stains and indicators
29
3.1.8.1 Gram stain
29
3.1. 8.2 Ethidium bromide stain
29
3.1. 8.3 Oxidase test Indicator 3.1. 8.4 Voges-Proskaur indicator
29
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29
3.1. 8.5 Methyl red indicator
30
3.1.9 Solution and buffers
30
3.1. 9.1 dNTPs Mixture
30
3.1. 9.2 10X PCR Buffer
30
3.1. 9.3 10X TBE Buffer
30
3.1. 9.4 Laoding dye 6X
30
3.1. 9.5 TE (Tris-EDTA) Buffer
30
3.1.10 RapID One System 20 Test :( Remel)
31
3.2Methods
33
3.2.1 Calculation of incidence rate
33
3.2.2 Sample collection
33
3.2.3 Laboratory identification of typhoid fever
34
3.2.3.1 Serological identification
34
3.2.3.1 Serological identification (Widal test)
34
3.2.3.2 White blood cell count
35
3.2.3.3 Isolation of Salmonella enterica Serovar Typhi
36
3.2.3.4 Gram stain
37
3.2.3.5 Biochemical test
38
3.2.3.5.1 Kligler iron agar
38
3.2.3.5.2 Urease test
38
3.2.3.5.3 Citrate test
38
3.2.3.5.4 Indol test
38
3.2.3.5.5 Methyl Red/Voges-Proskauer (MRVP) Tests
38
3.2.3.5.6 Motility test
39
3.2.3.5.7 Oxidase Test
39
3.2.3.6 Identification of Salmonella enterica serovar Typhi isolated by Remel RapID One System 3.2.4 Storage bacterial isolated
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39 40
3.2.5 Genomic DNA extraction
40
3.2.6 Polymerase Chain Reaction
41
3.2.7 Agarose gel electrophoresis
42
3.2.8 Statistical analysis CHAPTER FOUR:RESULTS& DISCUSSIONS
43 44-69
4.1 The incidence rate of Typhoid fever and paratyphoid fever
44
4.2 Widal test results
51
4.3 Isolation and primarily Identification of Salmonellae
57
4.4 Identification of salmonella using RapID One System
59
4.5 Identification by PCR for Salmonella isolates
61
Conclusions and recommendations
70-71
Conclusions
70
Recommendations
71
References Appendices
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72-92
LIST OF TABLES Table No. (1-1)
Title Kauffmann-White Classification of Salmonella
Page No. 12
taxa of medical Importance in developing Countries. (3-1)
Principles and component of the RapID One
31
System (4-1)
Widal test results represented as the number and
51
percentage of titer positive patients against O and H antigens of Salmonella enterica serovar Typhi among 120 clinically diagnosed typhoid patients and 15 healthy control persons (4-2)
Widal test results represented as the number and
55
percentage against O and H antigens of Salmonella enterica serovar Paratyphi B in 120 clinically diagnosed typhoid patients. (4-3)
RapID One System for (14) isolates of primarily
60
identified Salmonella enterica serovar Typhi (all with same reactions) and (11) isolates of Escherichia coli (all with same reactions). (4-4)
The amplification pattern of six Salmonella enterica serovar Typhi by using different primers, primarily identified by blood culture, biochemical tests and Rapid one system.
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68
LIST OF FIGURES Fig. No.
Title
Page No.
Figure (3-1)
Schematic diagram shows the identification
37
key of Salmonella enterica serovar Typhi and bacteria grown on biphasic blood culture.
Figure (4-1)
Total and sex specific incidence rate of typhoid
45
fever in Kalar district per 105 persons per year.
Figure (4-2)
Age group year’s specific incidence rate of
46
typhoid fever in Kalar district per 105 persons per year. Figure (4-3)
Incidence rate of typhoid fever according to
47
residency of the patients. Figure (4-4)
Total and sex specific incidence rate per 105
49
persons-year time paratyphoid fever in Kalar region Figure (4-5)
Average monthly typhoid fever incidence rate
50
per 105 populations in Kalar for the period 2005-2007. Figure (4-6)
Percentage of Widal test positive patients
52
according to sex group. Figure (4-7)
Comparative study of reinfections in male and
53
female in suspected cases of typhoid fever. Figure (4-8)
the percentage of bacterial isolates isolated from blood samples of 120 suspected typhoid patients identified primarily
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58
Figure (4-9)
Agarose gel electrophoresis of PCR products
62
for DNA amplification using the ConOriF/ConOriR primers for six identified primarily by RapID One system Figure (4-10) Agarose gel electrophoresis of PCR products
63
for DNA amplification using the hilAF / hilAR primers for six Salmonella enterica serovar Typhi identified primarily by RapID One system Figure (4-11) Agarose gel electrophoresis of PCR products
65
for DNA amplification using the ST1/ST2 primers for six Salmonella enterica serovar Typhi identified primarily by RapID One system Figure (4-12) Agarose gel electrophoresis of PCR products
66
for DNA amplification using the tyv-s/ tyv-as primers for six Salmonella enterica serovar Typhi identified primarily by RapID One system
Figure (4-13) Agarose gel electrophoresis of nested PCR products for DNA amplification using the ST3l, ST4 primers for six Salmonella enterica serovar Typhi PCR products amplified by ST1/ST2 primers.
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67
Introduction Enteric fever is a systemic disease, characterized by fever, abdominal pain and non-specific symptoms including nausea, vomiting, headache and anorexia (Vandepitte et al., 2003 and Maskey et al. 2006).When Enteric fever is caused by Salmonella enterica serovar Typhi, it is known as Typhoid fever and when due to serovars paratyphi A, B or C, it is called paratyphoid fever (Maskey et al., 2006).Of the three types, Salmonella paratyphi B is the most common (Ekdhal et al., 2005). Salmonella typhi, regarding the classification that Salmonella enterica serovar Typhi is mentioned as whenever it cited by old literature, lives only in humans, it is an obligate parasite. Persons with typhoid fever carry the bacteria in their bloodstream and intestinal tract. Typhoid fever spreads through contaminated food and water or through direct contact with someone who is infected (Buchwald and Blaster 1984; Pollack, 2003; CDC, 2005 and Women health –Guide, 2005). Transmission of Salmonella typhi has only been shown to occur by fecal-oral route (Pollack, 2003). Typhoid continues to be a significant global problem. There are an estimated 16.6 million cases and 600.000 deaths annually (Hart & Kariuki 1998; CDC, 2005 and WHO 2005) with most deaths occurring in Asia (440.000) and Africa (130.000), the majority of those affected are 3-19 years of age, with case fatality rates ranging from 0 to 5% (Pang et al., 1998). Typhoid and paratyphoid fever are contagious diseases. The incidence rate of Typhoid fever infection is higher than paratyphoid. In Typhoid fever, the infective dose is low (about 100 bacteria per person) while that of paratyphoid fever is higher (about 1000 bacteria per person) and subsequently food (where Salmonella bacteria can multiply) is implicated as the major vehicle for transmission (Ekdahl et al., 2005).
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The identification of most serotype Typhi infections, uses culture of the infectious agent from stool, urine, blood, bone marrow or bile (Wain et al., 2001) and the detection of Salmonella enterica serovar Typhi DNA is carried our by polymerase chain reaction (PCR) which has recently been shown to be a very sensitive index of infection(Massi et al.,2003). Widal test arises in O, H or Vi titers provides support for the diagnosis of typhoid fever (Shukla et al., 1997; Frimpong et al., 2000) , other serologic tests are being developed: ELISA (Nguyen et al., 1997 and House et al., 2005), salivary IgA (Herath 2003), a modified Widal test to detect IgM (Pai et al., 2003) and dipstick assay (Hatta et al., 2002). In Iraq, the incidence of typhoid fever reported has invariably been at a high level since the beginning of the 1990s. The cases diagnosed of whole country in 2000 and 2001 were 24614 and 21356 cases respectively, it needs to be pointed out that the death rate as a result of typhoid fever is particularly high and has reached the level of 10-20% (Korzeniewski, 2006). This study aimed at evaluating the incidence of typhoid fever and isolating and identifying of the causative agent Salmonella enterica serovar Typhi by serological, cultural and molecular methods in blood samples of clinically suspected typhoid patients in Garmiyan district.
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2. Literature review 2.1 A historical background In 1829, Louis Pierre offered the name typhoid fever to distinguish it from typhus disease as a separate and distinct disease, because of similar its in symptoms his views were published in the February number, American Journal, 1837 (Thomas, 1907). William Budd in 1856 studied typhoid outbreaks, and believed that typhoid was contagious and infectious agent (Chopra 1985). In 1880 typhoid bacillus was first observed by Eberth in spleen sections and mesenteric lymph nodes from a patient who died from typhoid. Robert Koch confirmed a related finding by Gaffky (1884) and succeeded in cultivating the bacterium in1881, but due to the lack of differential characters, separation of the typhoid bacillus from other enteric bacteria was uncertain (Reviewed by Todar, 2005). Salmon and Smith (1885) described a bacillus which was believed to cause hog cholerae. This bacillus now called Salmoella suis (Chopra 1985). Between 1896 and 1906, Mary Mallon worked as a cook in seven homes in New York city. Twenty-eight cases of Typhoid fever occurred in these homes while she worked in them. Examination of Mary's stool showed that she was shedding large numbers of typhoid bacteria though she exhibited no external symptoms of the disease. An article published in 1908 in the Journal of the American Medical Association referred to her as: Typhoid Mary: As a life time carrier, Mary Mallon was positively linked with 10 outbreaks of Typhoid fever, 53 cases, and 3 deaths (Prescott et al. 2005). At the beginning of the 19th century, typhoid was defined on the basis of clinical signs and symptoms and pathological (anatomical) changes. However, at that time, all sorts of enteric fevers were characterized as "typhoid" (Reviewed by Todar, 2005).
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Till (1990s) enteric fever was managed by cheap effective first line drugs. With the emergence of multidrug resistance typhoid the treatment has become complex with several antibiotics being used. This has further added to the problem of resistance with some strains showing resistance to both fluoroquinolones and cephalosporins (Kundu et al., 2006).
2.2 Typhoid fever 2.2.1 Pathogenicity Medical history attests that enteric fever has existed for several centuries. In 1659, Thomas Willis made the first description of the epidemic typhoid. In 1837, William Gerhard was the first who clearly differentiate typhoid from typhus fever (Alora and Lloren 1980). About 106 bacterial cells are needed to cause infection. Low gastric acidity – as in the elderly – can decrease the infective dose to 103 (Meltzer and Schwatz 2007). Salmonella enterica serovar Typhi has a combination of characteristics that make it an effective pathogen. This species contains a lipopolysaccharide macromolecular complex called endotoxin (Corales et al., 2004 and Brusch and Garvey, 2006). Once the bacteria are in the body, the incubation time is only about 8 to 48 hours. The disease results when the bacteria multiply and invade the intestinal mucosa, a phenomenon probably regulated by genes located in the “Salmonella Pathogenicity Islands” in the genome of Salmonella enterica serovar Typhi, where they produce an enterotoxin and a cytotoxin that destroy the epithelial cell (Khan, 2004 and Willey et al., 2009). The patient with typhoid fever suffers from a high fever of about 40˚C (104˚F) and continual headache. Diarrhea appears only during the second or third week, and the fever then trends to decline. In sever cases, which can be
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fatal, ulceration and perforation of the intestinal wall can occur (Tortora et al., 2007). At the sites of localization of S.typhi, the endotoxin of S.typhi induces macrophages to produce an array of cytokines, including tumor necrosis factor (TNF) and interferon, and various arachidonic acid metabolites. Cytokines alone, when acting locally at the sites of their production or when disseminated via the blood stream, can mediate the development of fever; the complications can be classified into: -those secondary to the local gastrointestinal lesions (e.g. hemorrhage and perforation) -those associated with toxemia (e.g.myocarditis, hepatic and bone marrow damage). - Those secondary to a prolonged serious illness. -those resulting from multiplication of the organisms in other sites, causing meningitis, osteomyelitis or endocarditis (Khan, 2004 and Mims et al., 2005). The macromolecular endoxin complex (LPS) consists of three components, an outer O-polysaccharide coat, a middle portion (the R core), and an inner lipid A coat (Giannella, 2004 and Prescott et al., 2005). Endotoxin, a typical of Gram negative organisms, as well as the Vi antigen are thought to increase virulence. It also produces and excretes a protein known as “invasion protein” that allows non-phagocytic cells to take up the bacterium, where it is able to live intracellularly. It is also able to inhibit the oxidative burst of leukocytes, making innate immune response ineffective (Pollack, 2003). Finally, with the Vi antigen, a polysaccharide capsule, Salmonella typhi and Salmonella paratyphi further protect themselves from lysis within the macrophage and from neutrophils and complement (Brusch and Garvey, 2006).
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2.2.3 Epidemiology Typhoid (enteric) fever is a systemic infection that is caused by Salmonella serotypes which are strictly adapted to humans or higher primates, including Salmonella enterica serovar Typhi, Paratyphi A, Paratyphi B and ParatyphiC. The disease is currently rare in the United States and Europe but endemic in Asia, Africa and Latin America, the Caribbean, and Oceania, but 80% of cases come from Bangladesh, China, India, Indonesia, Laos, Nepal, Pakistan, or Vietnam. Within those countries, typhoid fever is most common in underdeveloped areas, from where it can be imported by foreign travel and since 2004, small scale outbreaks have occurred in Kyrgyzstan, the Ukraine and Russia (Santos et al., 2001; Chau et al., 2007 and NaTHNaC,2007). It strikes about 12.5 million persons each Year (Women Health Guid, 2005). The World Health Organization (WHO) estimates that 16 to 33 million cases of typhoid fever occur each year, with 500,000 to 600,000 deaths (a case fatality rate of between 1.5 and 3.8%) (NaTHNaC, 2007). In many typhoid endemic areas, human immunodeficiency virus (HIV) infection is a serious public health concern. Although not reported from other endemic areas, a study in Peru has indicated that typhoid fever was 60 times more frequent in HIV-infected individuals as compared to the general population (Khan 2004). Paratyphoid fever is estimated to have caused an additional 5.4 million illness in 2000.The number is based on an estimated one case of Paratyphoid fever for every four cases of typhoid fever (Bhna et al., 2005). The damage caused by typhoid fever is reversible and limited if treatment is started early in the infection. This leads to a mortality rate of less than 1% among treated individuals who have an antibiotic-
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susceptible strain of S. typhi, making the outcome and prognosis for patients a positive one (Pollack, 2003). 2.2.3.1 Carriers About 1-3% of patients with enteric fever become chronic carriers. Patient usually continues to excrete recovery, which is defined as Salmonella typhi excretion in faces or urine for one year after infection. Chronic carriage is more common in women, in older patient and in those with underlying disease of the gallbladder (e.g. stones) or urinary bladder (e.g. Schistosomiasis) (Armstrong, 2001; Vandepitte et al., 2003 and Mims et al., 2005). However, some of them may remain as healthy carriers (Buchwald and Blaster 1984). The numbers of bacilli may vary widely from 500,000 to 450,000,000 per gram of faces (Chopra 1985). Carriers often have very high antibody titers to Vi antigen (WHO, 2000).
2.3 Salmonella enterica serovar Typhi According to Chopra 1985; Prescott et al., 2005 and Todar, 2005, most Salmonella strains shared the following characteristics: 1
Motile, Gram-negative bacteria
2
Lactose negative ;acid and gas from glucose, mannitol, maltose, and sorbitol; no acid from sucrose, salicin and lactose
3
ONPG test negative (lactose negative)
4
Indol test negative
5
Methyl red test positive
6
Voges-Proskauer test negative
7
Citrate positive(growth on Simmon's citrate agar)
8
Lysine decarboxylase positive
9
Urease negative
10
Ornithine decarboxylase positive
11
H2S produced from thiosulfate
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12
Do not grow with KCN
13
Phenylalanine and tryptophan deaminase negative
14
Gelatin hydrolysis negative
15
% G+C 50-53 Salmonella enterica serovar Typhi is gram-negative, facultative
anaerobic, non-capsulated and flagellated bacilli in the family of Enterobactericeae. They are motile bacilli 1-3 µm, in length and 0.5-0.7 µm in thickness (WHO, 2000; Hukstep et al., 2002; Lee, 2002 and Crum, 2003).
2.3.1 Taxonomy The names given to salmonellae do not follow the usual rules of nomenclature because of their importance in pathology (Krieg, N.R., 1984). The current taxonomy and nomenclature of the genus Salmonella proposed in the 1980s cited by Le Minor and Popoff (1987) has received wide acceptance, although it does not conform to the rules of the Bacteriological Code. The taxonomic opinion of Le Minor and Popoff (1987) and Reeves et al. (1989), that the genus Salmonella should currently comprise two species and that the type species Salmonella enterica should be divided into six subspecies, results in the taxonomy and nomenclature currently in use by the WHO and other organizations (Tindall et al., 2005). Based on iso-enzymes, rRNA sequences, and DNA hybridization, microbial taxonomists consider almost all of the Salmonella that infect mammals and birds to be one species Salmonella enterica (Fierer and Guiney 2001). The classification of Samonella is complex because the organisms are a continuum rather than a defined species. Much of the confusion was due to the practice of naming serotypes (serovars) and
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equating them with species. All phylogenetic data indicate that the evolution of the genus Salmonella is a continuum (Garrity et al., 2004). The members of the genus Salmonella are grouped according to the classification on the basis of epidemiology, host range, biochemical reaction and structure of the O, H and Vi antigens. DNA-DNA hypridization studies have demonstrated that there are seven evolutionary groups (Brooks et al., 2001 and Brooks et al., 2007). There are more than 2500 serotypes of salmonellae, including more than 1400 in DNA hybridization group I that can infect humans. Salmonella are grouped based on the somatic O antigen and further divided into serotypes based on flagellar H and surface virulence (Vi) antigens based on DNA studies. The species name Salmonella enterica have been widely accepted, and the organisms in DNA hybridization group I are Salmonella enterica subspecies enterica. The organisms, in the other groups, have other subspecies names. It seems probable that the widely accepted nomenclature for classification (Brooks et al., 2007). Salmonellae are now considered one of two species: Salmonella enterica (formerly called Salmonella Choleraesuis) and Salmonella bongori. Salmonella enterica has 6 subspecies (l,llla,lllb,lV,Vl); Salmonella bongori
has one (V).
Salmonella typhi and Salmonella paratyphi are Salmonella enterica l subspecies, serotypes typhi and paratyphi (Garrity et al., 2004; Brusch and Garvey, 2006). Now Salmonella enterica serovar Typhi classified as follows (Garrity et al., 2005): Domain
Bacteria
Phylum
Proteobacteria
Class
Gammaproteobacteria
Order
Enterobacteriales
Family
Enterobacteriaceae
Genuse
Salmonella enterica serovar Typhi
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2.3.2 Antigenic structure The genus Salmonella has three kinds of major antigens identifying applications: somatic, surface, and flagellar (Todar, 2005).
2.3.2.1
Somatic(O) antigens
The O antigen is the somatic antigen of Salmonella enterica serovar Typhi and is shared by salmonella enterica serovar Paratyphi A, Paratyphi B, other salmonella species and other members of the Enterobactericeae family (Rodrigues, 2003). Somatic antigens are heat stable and alcohol resistant and they are used for serological identification (Todar, 2005), and associated with a change from smooth to rough colony form (Brooks et al., 2001). O antigens are parts of the surface of the outer membrane, the Lipopoly saccharide (LPS) are determined by specific sugar sequence on the LPS and the LPS composed of four to six sugars depending on the O antigen (Perch et al., 2003 and Giannella, 2004). Salmonella O Antigens are of two types: O Group antigens and ancillary antigens; O Group antigens are most important for determining serotypes. Ancillary O antigens typically encoded by extra-chromosomal elements (bacteriophages, plasmids) found in specific O groups and mostly can vary within a given serotype, so are less important for serotype determination (CDC, 2006). Salmonella are grouped by their O antigens (Table 1-1). Each group has what is called a group factor (Cheesbrough, 1984 and Perch et al., 2003). Antibodies against the O antigens are predominantly IgM, risen early in the illness and disappear early (Corales et al., 2004).
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2.3.2.2
Surface (Envelop) antigens
Surface antigens commonly observed in other genera of enteric bacteria (e.g Escherichia coli and Klebsiella), and may be found in some Salmonella serovars (Cheesbrough, 1984 and Chopra 1985). Vi antigen is a glycolipid , heat liable, poorly immunogenic and only low titer of antibodies are produced ,detection of Vi antibody is not helpful for diagnosis of cases of typhoid fever, the persistence of Vi antibody indicates the development of the carrier state (Chopra 1985). Surface antigens in Salmonella may mask O antigens, and the bacteria will not agglutinate with O antisera. The Vi antigen occurs in only three Salmonella serovars (Out of about 2,200) which are Typhi, Paratyphi C, and Dublin. Strains of these three serovars may or may not have the Vi antigen (Todar, 2005). It is associated with virulence and can be detected using Vi antiserum (Cheesbrough, 1984).
2.3.2.3 Flagellar (H) antigens H antigens are the filamentous portion of the bacterial flagella; H antigen is made up of protein subunits called flagellin. The ends of flagellin are conserved and give the filament its characteristic structure. The antigenically variable portion of flagellin is the middle region of the protein, which is surface-exposed heat –stable proteins (Perch et al., 2003). Mixing salmonella cells with flagella specific antisera gives a characteristic pattern of agglutination (bacteria are loosely attached to each other by their flagella and can be dissociated by shaking), although antiflagellar antibodies can immobilize bacteria with corresponding H antigens, a few Salmonella enterica serovars (e.g.,Enteritidis,Typhi) produce flagella which always have the same antigenic specifity, such an H antigen is then called monophasic. Most Salmonella serovars, however, can alternatively produce flagella with two different H antigenic
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specificties, then they are called diphasic (Todar, 2005), so that Salmonella are serotyped by their H antigens (Cheesbrough, 1984). Table (1-1) Kauffmann-White Classification of Salmonella taxa of Medical Importance in Developing Countries. The O antigen in bold type is common to all members of the group (Cheesbrough, 1984).
SALMONELLA
O Antigens
H Antigens Phase1
phase2
GroupA: S.paratyphi A
1,2,12
a
S.paratyphi B
1,4,5,12
b
S.derby
1,4,5,12
f,g
S.typhimurium
1,4,5,12
i
1,2
S.heidelberg
(1)*,4,5,(12)*
r
1,2
S.choleraesuis
6.7
c
1,5
S.paratyphiC
6.7.(Vi)*
c
1,5
S.oranienburg
6.7
m,t
S.garoli
6.7
i
1,6
S.thompson
6.7
k
1,5
S.bareilly
6.7
y
1,5
S.typhi
9,12,(Vi)*
d
_
S.enteritidis
1,9,12
g,m
_
-
Group B: 1,2 (1,2)*
GroupC:
--
GroupD:
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S.pullorum-gallinarum
1,9,12
--
_
(non motile) GroupE: S.weltevreden
3,10
r
z6
S.anatum
3,10
e,h
S.durham
13,23
b
enz15
S.wanhington
1,13,23
z
1,w
S.cubana
1,13,23
z29
1,6
Group G:
(*) indicates that the antigen may be present or absent.
2.4 Identification methods Salmonella enterica serovar Typhi, the causative agent of typhoid fever is identified by serological, cultural and by molecular methods.
2.4.1 Serological methods A serological method relies classically on the demonstration of a rising titer of antibodies and such rising may not be detected even for cases confirmed as typhoid fever by blood culture (Parry, 1999). Salmonella spp. are characterized according to their O and H antigens and by grouping them depending on Kauffman-White scheme (Baron et al., 1994). Ritually, the provisional laboratory identification of infection by Salmonella spp. is the serological test called Widal test, then confirmed by culture methods or even the detection of the presence of bacteria in blood by gene amplification (PCR).
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_
2.4.1.1 Widal test In 1880, Eberth discovered the typhoid agglutinins and its diagnostic application (Alora and Lloren 1980). Widal test is a tube agglutination test employed in the serological diagnosis of enteric fever. The test is named in 1896 after Georges Fernand Isidore Widal. He detects agglutinating antibodies against the O and H antigens of Salmonella typhi and Salmonella Paratyphi A, B and C (Berger, 1999; Olopenia&King, 2000 and Rao, 2009). The Widal test is reported by giving the titer for both O and H antibodies. In acute infection, O antibody appears first, rising progressively, later falling, and often disappearing within a few months. H antibody appears slightly but persist longer (Corales et al., 2004). The cutoff for positively chosen in a particular community depends on the background level of typhoid fever (Clegg et al., 1994). The titers of H or O antibodies, or both agglutinins are significantly raised ( i.e. titers greater than 1 in 180 or 1 in 200 depending on the titers found in local healthy people) (Cheesbrough, 1984).
2.4.1.2 Enzyme linked immunosorbent assay (ELISA) Enzyme linked immunosorbent assay (ELISA), ELISA has become one of the most widely used serological tests for antibody or antigen detection; it was first developed during the 1960s. The basic test consists of antibodies bonded to enzymes; the enzymes can catalyze the reaction of many substrate molecules, greatly amplifying the reaction and enhancing detection by formation of a colored end product and allow direct observation of the reaction or by automated spectrophotometric reading (Forbes et al., 2007). Two basic methods are used: The double antibody sandwich assay and the indirect immunosorbent assay (Prescott et al, 2005).
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ELISA for detection of Salmonella causing typhoid also called Enzyme
immunoassay
(EIA)
or
Typhidot
test:
Adot
enzyme
immunoassay that detects IgG and IgM antibodies against a 50 KD outer membrane protein distinct from the somatic (O), flagellar (H) or capsular (Vi) antigen of Salmonella typhi is commercially available as Typhidot (Casem et al., 2002).The sensitivity and specificity of this test has been reported to vary from 70-100% and 43-90% respectively (Choo et al., 1999). The detection of IgM reveals acute typhoid in the early phase of infection, while the detection of both IgG and IgM suggests acute typhoid in the middle phase of infection. In areas of highly endemicity where the rate of typhoid transmission is high, the detection of specific IgG increases. Since IgG can persist for more that 2 years after typhoid infection, the detection of IgG can not differentiate between acute and convalescent cases (Saha et al., 1999).
2.4.1.3 Latex agglutination test Rapid latex agglutination test has also been developed to detect specific antigens in the culture supernatants. Its main utility is in rapid identification of species of Salmonella (Snigh, 2001). Serological grouping on isolates of Salmonella using commercially a viable polyvalent antisera designated A,B,C,C2,D,E,F,G,H, and Vi (Baron, et al.1994 and Forbes et al.,2007). The following antisera are required to identify Salmonella typhi Salmonella O antiserum Factor 9 (GroupD) Salmonella H antiserum d Salmonella Vi antiserum (Cheesbrough, 1984)
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2.4.2Culture Identification of Salmonella may sometimes pose a problem, because some strains vary in their biochemical reactions and may even share antigens with other Gram-negative organisms (Vandepitte et al., 2003). Salmonella cultures take 4–7 days for isolation and identification (Jimenez and Castro, 2004). Culture methods for diagnosis of typhoid fever is by demonstration of typhoid bacilli Salmonella typhi in the blood, urine, or stool (Prescott et al., 2005).
2.4.2.1 Blood cultures It was reported that blood culture is with higher specificity and helpful in diagnosis of early typhoid while its sensitivity is poor, Salmonella typhi can be detected only for 40-45% of patients and it has the promise of diagnosis in first (Guerra- Caceras et al., 1979 and Werner et al., 1976). Clot culture is also used, but when the inhibitory effect of serum is obviated, it has not been found to be of superior sensitivity as compared to blood cultures, in spite of that, clot culture gives higher rate of isolation of Salmonella than blood cultures (Hoffman et al., 1986). Salmonella can be easily cultured in most microbiologic laboratories with use of routine culture media (Blood agar and Macconkey agar) (Wain et al., 2001).
2.4.2.2 Stool cultures The findings of stool cultures are usually positive in approximately 40-50% of patients with typhoid fever during the second week, and from about 80% of patients during the third week (Cheesbrough 1984). Specimen should preferably be processed within 2 hours after collection. If there is a delay the specimen should be stored in a refrigerator at 4˚C in a cool box with freezer packs. The sensitivity of stool culture depends on
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the amount of feces cultured, and the positively rate increases with the duration of the illness (Parry et al., 2002).
2.4.2.3 Bone marrow cultures Salmonella typhi is an intracellular pathogen in the reticuloendothelial cells of the body including the born marrow. Studies have revealed that the median bacterimia in the bone marrow is 9 CFU/ml compared to 0.3 CFU/mL in blood (Wain et al., 2001). The overall sensitivity of bone marrow cultures ranges from 80-95% and is good even in late disease and despite prior antibiotic therapy (Parry et al., 2002). The invasive nature of bone marrow aspiration deters from its use as a first line investigation for diagnosis of typhoid fever (Kundu et al., 2006).
2.4.2.4 Urine and other cultures In 1901, Sir William Osler, isolated typhoid bacilli from urine (Berger, 1999). Urine culture is positive for Salmonella typhi for a quarter to one third of cases, but there seems to be no regular excretion pattern in the urine. The urine is free from organism's weeks before the faeces become negative (World health organization, 2006). A single rectal swab culture at hospital admission can be expected in 30-40% of patients (Corales et al., 2004).
Rectal swab should be avoided as these are less successful
(Parry et al., 2002). A sensitivity of 63% has also been reported from culturing skin snips of rose spots. Salmonella typhi has been isolated from the cerebrospinal fluid, peritoneal fluid, mesenteric lymph nodes, resected intestine, pharynx, tonsils, abscess, bone, and urine among others (Corales et al., 2004).
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2.4.3 Molecular identification 2.4.3.1 Identification by Polymerase Chain Reaction (PCR) Between 1983 and 1985, Kary Mullis developed a technique to synthesize large quantities of a DNA fragment without cloning it (Prescott et al., 2005). PCR as a diagnostic modality for typhoid fever was first evaluated in 1993 when Song et al. successfully amplified the flagellin gene of Salmonella typhi in all cases of culture proven typhoid fever and from none of the healthy controls. Moreover, some patients with culture negative typhoid fever were PCR positive suggesting that PCR diagnosis of typhoid may have superior sensitivity than culture, that it detects bacteria in blood even with the antibiotic treatment (Nagarajan,et al.,2009). Over the next 10 years, a handful of studies have reported PCR methods targeting the flagellin gene, somatic gene, Vi antigen gene, 5S23S spacer region of the ribosomal RNA gene, invA gene and hilA gene of Salmonella typhi for diagnosis of typhoid fever (Castro and Jimenez, 2004; Prakash et al., 2005). By PCR, 1-5 bacteria/ml are detectable and results can be available in 1-2 days (Hashimoto et al., 1995 and Haque et al., 1999). Song and Coworkers described a nested PCR for the detection of Salmonella typhi DNA in blood specimens (Song et al., 1993). A nested PCR strategy could offer a useful tool for rapidly and specifically detection Salmonella typhi from clinical specimens (Hashimoto et al., 1995), but is relatively complicated to perform and is not suitable for use in routine clinical practice in laboratories in the countries where typhoid fever is endemic. Also, in countries where Salmonella paratyphi is a common cause of enteric fever, this method will not be useful (Hatta and Smits, 2007).
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2.5 Treatment James Currie (1756–1805), a Scot, was the first to use cold baths in treatment of typhoid fever; and by monitoring the patient’s temperature using a thermometer, he was able to adjust the temperature and frequency of the baths to treat individual patients (Berger, 1999). In 1948, Woodward and his groups were they first used Chloramphenicol against typhoid fever (Alora and Lloren 1980). The rediscovery of oral rehydration therapy in the 1960s, and proven in the Bangladesh Liberation War, provided a simple way to prevent many of the deaths of diarrheal diseases in general. Typhoid fever in most cases is not fatal. Antibiotics, such as ampicillin, chloramphenicol, trimethoprim-sulfamethoxazole, Amoxicillin and ciprofloxacin, have been commonly used to treat typhoid fever in developed countries. Prompt treatment of the disease with antibiotics reduces the case-fatality rate to approximately 1% (Parry and Beeching 2009 and Thaver et al., 2008). Many text books suggest that the case fatality rate has been less than 2% after the introduction of chloramphenicol (Worku, 2000). Treatment with ceftriaxone, trimethoprim-sulphame – thoxazole, or ampicillin has reduced the mortality rate to less than 1 % (Prescott et al., 2005). In the 1990s, resistance to nalidixic acid was reported in Southeast Asia. This resistance, most commonly caused by point mutations in the gyrA and parC genes, spread rapidly, and now its distribution is worldwide (Shakespeare et al., 2005). Standard treatment with chloramphenicol or Amoxicillin is associated with a replace rate of 5-15% or 4-8% respectively, whereas the newer quinolones and third generation cephalosporins are associated with higher cure rates (WHO 2003).
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Treatment of typhoid fever is the possibility of toxic reactions, first described by Reilly in the late 1940s. These reactions are thought to be due to a sudden release of endotoxin in mesenteric lymphatic nodes by bacteria lysed by antibiotics theraby. Being neurotropic, the endotoxin can cause neurologic symptoms, cardiovascular collapse, and temperature deregulation. These reactions are mainly seen in patients treated with chloramphenicol or amoxicillin, usually within 24 hours after the start therapy .Toxic reactions (hypothermia) occurred in 9 patients treated with oral chloramphenicol during an outbreak of 330 cases of enteric fever in Egypt. Hypothermia has been reported by others, and attributed to chloramphenicol hypersensitivity, but also consists of a sudden release of endotoxin from killed organisms (Caumes et al., 2001). Chloramphenicol resistance started to develop within two years of the drug introduction and until 1972 chloramphenicol resistant Salmonella typhi became a major problem. Outbreaks of chloramphenicol resistant salmonella occurred in Mexico, India, Vietnam, Thailand, Korea and Peru. These strains were also found to be resistant to sulfonamide, tetracycline and streptomycin. Amoxicillin and trimethoprim-sulphame- thoxazole were effective alternatives till the end of 1990’s when strains resistant to all the first line anti-Salmonella drugs used at that time, were reported. Multi-drug resistant Salmonella typhi’s (MDRST) resistance rarely develops during course of treatment but instead results from clonal dissemination of individual multi-drug resistant Salmonella typhi or from transfer of R-plasmid (Mirza et al., 2000; Shananhan et al., 1998 and Mushtaq 2006). Adachi et al. (2005) strongly suggested that high level of quinolone resistance was induced through the long term carrier state of Salmonella paratyphiA under selective pressure of frequent quinolone administration. Chloramphenicol binds to 50S bacterial- ribosomal subunits and inhibits
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bacterial growth by inhibiting protein synthesis. Amoxicillin interferes with synthesis of cell wall mucopeptides during active multiplication resulting
in
bactericidal
activity
against
susceptible
bacteria.
Trimethoprime and sulfamethoxazole inhibits bacterial growth by inhibiting synthesis of dihydro folic acid (Corales et al., 2004).
2.6 Vaccination In 1897, Almroth Edward Wright developed an effective vaccine. In 1909, Frederick F. Russell, a US army physician, developed an American typhoid vaccine and two years later his vaccination program became the first in which an entire army was immunized. It eliminated typhoid as a significant cause of morbidity and mortality in the US military. A vaccine against typhoid fever was developed during World War II by Ralph Walter Graystone Wyckoff (Wyckoff 1995 and WHO 2007). There are two vaccines currently recommended by the World Health Organization (2000) for the prevention of typhoid: these are the injectable Typhoid polysaccharide vaccine (sold as Typhim Vi by Sanofi Pasteur and Typherix by GlaxoSmithKline) contains fragments of inactivated Salmonella typhi bacteria. It works by stimulating the body's immune response to the typhoid bacteria, without actually causing the disease (Easmon, 2009). Both are between 50% to 80% protective and are recommended for travelers to areas where typhoid is endemic (WHO 2008). The live and oral Ty21a vaccine (sold as Vivotif Berna) is a live attenuated strain of Salmonella typhi Ty21a that was developed in the early 1970s by chemical mutagenesis (World Health Organization, 2003 and WHO 2008). Both vaccines are equally effective and offer 65-75% protection against the disease (CBWInfo. 1999). Ty21a vaccine can
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induce cellular immune responses lasting for at least 2 Years after immunization (Salerno et al., 2002). Among available vaccines, the oral live attenuated vaccine and the IM Vi capsular polysaccharide vaccine have comparable protection rates (50-80%). Vaccination cannot substitute for hygienic food and water practices (Hopkins, 2007).
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Materials and Methods 3.1 Materials 3.1.1 Apparatus and Equipments
No. Name
Manufacture Company
Country
1.
Incubator
LabTec
Korea
2.
Autoclave
LabTec
Korea
3.
Shaking water Bath
LabTec
Korea
4.
Auto Vortex
Stuart Scientific
UK
5.
Eppendorf centrifuge
Eppendorf
Germany
6.
Sensitive Balance
Adam
AFA
7.
Micropipette(different
Slamed and
Germany
sizes)
Eppendorf
Electrophoresis Power
Aplex
France
CrealCon Technologies
The
8.
supply& Horizontal Gel Tank 9.
Thermocycler
Netherlands 10.
UV Viewing Cabinet
Bijing Linyi
China
11.
Camera
Sony
Japan
12.
Ice Maker
LabTec
Korea
13.
Microwave
LG
Korea
14.
Hotplate Stirrer
LabTec
Korea
15.
Refrigerator
Hitachi
Japan
16.
Water Distillater
LabTec
Korea
17.
pH meter
WTW
Germany
18.
Oven
LabTec
Korea
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19
Clean Bench
LabTec
Korea
20.
Compound Light
Olympus
Japan
Microscope 21.
Ultracentrifuge
Sigma
USA
22.
Chest Freezer
Shownic
Malaysia
23.
Centrifuge
Truip International Corp Korea
24.
Haemocytometer
Thoma
Germany
3.1.2 Chemicals
No.
Name
Manufacture Company
Country
1.
Agarose
Fermentas
Germany
2.
Chloroform
α-Alpha
India
3.
Ethanol (99.9%)
GCC
UK
4.
Ethidium Bromide
Sigma
Germany
5.
Glycerol
Fisong
England
6.
Normal saline
ADWIK
Egypt
7.
Oil immersion
BDH
England
8.
TBE Buffer
Fermentas
Germany
9.
Loading dye
Fermentas
Germany
10. Glacial acetic acid
BDH
England
11. HCl
Fluka
Switzerland
12. NaCl
Fluka
Switzerland
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3.1.3Culture Media No.
Culture Media
Company
1 Brain heart infusion broth
HiMedia(India)
2 Brain heart infusion agar
HiMedia(India)
3 Agar agar
HiMedia(India)
4 Nutrient broth
HiMedia(India)
5 Nutrient agar
HiMedia(India)
6 Blood agar
HiMedia(India)
7 MacConkey agar
HiMedia(India)
8 Salmonella Shigella agar
HiMedia(India)
9 Simmon citrate agar
HiMedia(India)
10 Urea agar
HiMedia(India)
11 Peptone Water
LabM (UK)
12 Kligler iron agar
HiMedia(India)
13 Kovacs Indol Reagent
HiMedia(India)
14 Tryptone
HiMedia(India)
15 Yeast extract
HiMedia(India)
3.1.4 Kits and Enzymes No.
Enzymes and Kits
Company
1
Taq DNA Polymerase
Fermentas (Germany)
2
Gram stain Kits
Crescent Diagnosis(KSA)
3
Rapid One System20 Test
Remel (USA)
4
Genomic
DNA
Purification
Kit Fermentas (Germany)
Consisting of :Precipitation Solutions, Lysis solution and 1.2M NaCl 5
100bp DNA Ladder marker
Fermentas (Germany)
6
Mgcl2
Fermentas (Germany)
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3.1.5 Primers Gene
primers
Sequences
Fragment
Reference
amplicon (bp) H1-d
H1-d
Rfbe(tyv)
hilA
OriC
ST1
5'TATGCCGCTACATATGATGAG-3'
ST2
5'-TTAACGCAGTAAAGAGAG-3'
ST3
5'- ACTGCTAAAACCACTACT-3'
ST4
5'-TGGAGACTTCGGTCGCGTAG-3'
tyv-s
5'-GAG GAA GGG AAA TGA AGC TTT T-3'
tyv-as
5'-TAG CAA ACT GTC TCC CAC CAT AC-3'
US
5'-CGGAACGTTATTTGCGCCATGCTGAGGTAG-3'
DS
5'- GCATGGATCCCCGCCGGCGAGATTGTG-3'
ConOri-F
5'-GCGGTGGATTCTACTCAAC-3'
ConO ri-R
5'-AGAAGCGGAACTGAAAGGC-3'
495
Frankel 1994.
363
Frankel 1994.
615
Hirose et al., 2002
784
Castro et al., 2002
461
Woods et al.,2008
3.1.6 Widal test kit Febril antigen kit (Plasmatec, UK) contained the following Salmonella antigens: 1. Salmonella typhi H antigen 2. Salmonella typhiO antigen 3. Salmonella typhi A-H antigen 4. Salmonella typhi B-H antigen 5. Salmonella typhi C-H antigen 5. Salmonella typhi A-O antigen 6. Salmonella typhi B-O antigen. 7. Salmonella typhi C-O antigen.
3.1.7 Culture Media 3.1.7.1. Brain heart infusion broth The medium was prepared by dissolving 37 gm of the powder (Himedia, India) in 1litter of distilled water and autoclaved. Then, it was distributed into 30 ml screw cap bottles.
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3.1.7.2. Brain heart infusion agar The medium was prepared by dissolving 52 gm of the powder (Himedia, India) in 1litter of distilled water, dissolved by heating and stirring then autoclaved. 3.1.7.3 Biphasic blood culture It was prepared according to (Himedia, India) company by pouring 7 ml of autoclaved brain heart infusion agar in 30 ml screw cap bottles and prepared as slants, then 20 ml of autoclaved brain heart infusion broth were added. To prepare blood culture at the time of using, 2 ml of blood were added. 3. 1.7.4 Blood agar It was prepared by dissolving 40 gm of the blood agar base (Himedia, India) in 1litter of distilled water and then autoclaved; human blood (7%) was added after cooling to 50˚C, according to the manufacturer. 3. 1.7.5 MacConkey′s agar It was prepared by dissolving 51.5 gm of the powder (Himedia, India) in 1litter of distilled water and autoclaved, according to manufacturer. 3. 1.7.6 Salmonella Shigella agar It was prepared by dissolving 63 gm of the powder (Himedia, India) in 1litter of distilled water and autoclaved, according to manufacturer. 3. 1.7.7 Kligler iron agar It was prepared by dissolving 57.5 gm (Himedia, India) in 1litter of distilled water and autoclaved, according to manufacturer. It contains glucose, lactose, and phenol red. When this medium is used in slants, it is an excellent medium for detecting glucose and lactose fermentation and it’s contains Iron salts that react with H2S to form a dark precipitate of Iron sulfide (Aneja, 2003 and Brown, 2005).
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3. 1.7.8 Simmon′s Citrate agar It was prepared by dissolving 24.28 gm (Himedia, India) in 1litter of distilled water, autoclave, according to manufacturer. This test was used to determine the ability of an organism to utilize sodium citrate as carbon source and inorganic ammonium salts as nitrogen source. Bacteria that can grow on this medium turn the green indicator to blue (Aneja, 2003). 3. 1.7.9 Indol Broth It was prepared by dissolving 15gm from peptone water (LabM, UK) in 1litter of distilled water, according to manufacturer, then sterilized by autoclave. This test was used to determine the ability of an organism to split tryptophan to form compound indol, pyruvic acid and ammonia. Positive result changes pink colored ring after addition Kovac's reagent (Forbes, 2007). 3. 1.7.10 Semi solid agar for motility test (Brown, 2005 and Forbes, 2007) It was prepared by adding 0.5 gm from agar agar (Himedia, India) to 100 ml Nutrient broth (Himedia, India), and the pH was adjusted to 7.4 and then autoclaved. 3. 1.7.11 Methyl Red-Voges Proskauer broth This test is used to determine the ability of an organism to produce and maintain stable acid end products from glucose fermentation (Aneja, 2003; Brown, 2005 and Forbes, 2007). 3. 1.7.12 Nutrient broth It was prepared by dissolving 13gm from the powder (Himedia, India) in 1litter of distilled water and autoclave, according to manufacturer. 3. 1.7.13 Urea agar base It was prepared by dissolving 24gm from the powder (Himedia, India) in 950 ml of distilled water, autoclave, according to manufacturer.
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This test was used to determine the ability of an organism to produce Urease enzyme (Aneja, 2003 and Brown, 2005). 3.1.7.14 LB media It was prepared (Fermentase, Germany) by dissolving 10 gm of tryptone , 5gm Nacl and 5gm yeast extract in 1 litter distalled water and the pH was adjusted to 7.0 then autoclaved.
3.1.8 Stains and indicators 3.1.8.1 Gram stain Gram stain solution was prepared according to Crescent diagnosis company, (KSA). It composed of Crystal violet, Iodine solution, acidic alcohol and Safranine. 3. 1.8.2 Ethidium bromide stain It was prepared by dissolving 0.1gm of ethidium bromide powder in 1ml distilled water (Brown, 2001). 3. 1.8.3 Oxidase test Indicator Is to determine the presence of bacterial cytochrom oxidase. It was prepared as 1% by dissolving 0.05 gm of tetra methyl-p- phenlyene diamine dihydrochloride in 5 ml distilled water (Brown, 2005). 3. 1.8.4 Voges-Proskaur indicator The indicator consisted of two solutions: Solution A: It was prepared by dissolving 40 gm of KOH in 100ml of distilled water. Solution B: It was prepared by dissolving 5 gm alpha-Naphthol in 100 ml absolute Ethanol (Brown, 2005).
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3. 1.8.5 Methyl red indicator It was prepared by dissolving 0.1 gm of Methyl Red indicator in 300 ml of 95% Ethanol ,the volume was completed to 500ml distilled Water (Aneja, 2003 and Brown, 2005).
3.1.9 Solutions and Buffers 3. 1.9.1 dNTPs Mixture This was provided by Fermentas(Germany)with concentration of 10mM . 3. 1.9.2 10X PCR Buffer This Buffer was provided by Fermentas (Germany) consisting of Taq buffer pluse KCl. 3. 1.9.3 10X TBE Buffer This Buffer was provided by Fermentas (Germany) consisting of 108gm Tris-base, 55 gm Boric acid and 0.5M EDTA (PH 8)40 ml in 1 liter Distill Water. 3. 1.9.4 Laoding dye 6X Laoding dye 6X which was provided by Fermentas (Germany) consisting of loading dye and SDS solution. 3.1.9.5 TE (Tris-EDTA) Buffer The Buffer 10X was consisted, according to Fermentas company (Germany), of 1M Tris base (pH 8)10ml and 0.5M EDTA (pH 8) 2ml in 1liter double distilled water. Buffer 1X was prepared as follows: - 10 mM Tris base (pH 8) was prepared by dissolving 1.2 gm in 10 ml. -
1mM EDTA (pH 8) was prepared by dissolving 0.29 gm in 2 ml. 10 ml of Tris base and 2 ml of EDTA were completed to 1 litter by double distilled water.
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3.1.10 RapID One System 20 Test (Remel, USA) Remel RapID One System is a qualitative micromethod employing conventional and chromogenic substrates for the identification of medically important Enterobacteriaceae and other selected Oxidase negative bacilli isolated from human clinical specimens. The RapID One System is comprised of RapID One Panels and RapID One Reagent. Each RapID One Panels has several reaction cavities molded into the periphery of a plastic disposable tray. Test organism suspension was prepared by adding bacteria colonies in MacConkeys agar for 37˚C at 24 hours in Rapid inoculum fluid (2ml) to obtain suspension concentration equivalent to MacFarland standared 2#. The suspension was used to rehydrate cavities of the tray. Each RapID tray contains reactive ingredient and the results was interpretated as shown in table (3-1).
Table (3-1) Principles and Component of the RapID One System Cavity
Test
Reactive Ingredient
Interpretation result
Code +ve
-ve
1
URE
Urea
Red or violet
Yellow or orange
2
ADH
Arginine
Bright purple
Yellow,gray,or
or blue
yellow-green
Bright purple
Yellow,gray,or
or blue
yellow-green
Bright purple
Yellow,gray,or
or blue
yellow-green
3
4
ODC
LDC
Ornithine
Lysine
5
TET
Aliphatic thiol
Yellow
Red or orange
6
LIP
Fatty acid ester
Yellow
Red or orange
7
KSF
Sugar aldehyde
Yellow
Red or orange
8
SBL
Sorbitol
Yellow
Red or orange
9
GUR
p-Nitrophenyl-B,D-
Yellow
Clear or tan
glucuronide 10
ONPG
o-Nitrophenyl-B,D-galactoside
Yellow
Clear or tan
11
BGLU
p-Nitrophenyl-B,D-glucoside
Yellow
Clear or tan
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12
BXYL
p-Nitrophenyl-B,D-Xyloside
Yellow
Clear or tan
13
NAG
p-Nitrophenyl-n-acetyl-B,D-
Yellow
Clear or tan
Red
Yellow or orange
glucosaminide 14
MAL
Malonate
15
PRO
Proline-B-naphthylamide
Violet, purple,red, or
Clear, Yellow, orange, or very pale pink
dark pink 16
GGT
y-Glutamyl-B- naphthylamide
Violet, purple,red, or
Clear, Yellow, orange, or very pale pink
dark pink 17
PYR
Pyrrolidonyl-B naphthylamide
Violet, purple,red, or
Clear,yellow,orange,or very pale pink
dark pink 18
ADON
Adonitol
Yellow or very light orange
18
IND
Tryptophane
Brown,
Red or dark red orange Orange or red
black,or purple
RapID One Reagent was supplied by Remel which were: • RapID One Reagent(15ml/Btl) Reactive ingredient per later: p-Dimmethylaminocinnamalehyde 0.06gm • RapID Inoculation Fluid (2ml/Tube) KCl
6gm
CaCl2
0.5 gm
Demineralized Water
1000ml
• RapID Spot Indole Reagent (15ml/Btl) p-Dimmethylaminocinnamalehyde
10gm
Hydrochloric Acid
100ml
Demineralized Water
900ml
The resulting patterns of positive and negative test scores were used as the basis for identification of the isolates by comparison of test
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results to reactivity patterns stored in a database, according to Remel Company.
3.2 Methods 3.2.1 Calculation of Incidence Rate Incidence rate was calculated per 100.000 persons per Year using the following formula: Ra=
na T*PA Where:
(Judith et al., 2000)
Ra= the total or sex specific group incidence rate. na=the number of events in the total and sex group during the time interval. T= length of the time interval. Pa= average size of the population in the sex group or total during the time. The Mortality rate can be calculated by using the formula: Mortality rate = number of deaths due to a given disease Size of the total population with the same disease (Prescott et al., 2005)
3.2.2 Sample collection One hundred and twenty blood samples were collected from equivalent number of clinically diagnosed to be suspected as typhoid patients in Kalar General Hospital in the period between September 2008 to May 2009, all were inpatients except one was outpatient. Patients given a designation (P) followed by a serial number for each patient (P1, P2------P120). Blood was also collected from healthy persons (as they had no signs of typhoid and negative for Widal test) as control. Five milliters of venous blood were taken aseptically.
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Using a 23G gauge syringe from each subject, then divided into three parts,
one part (2 ml) was used to inoculate biphasic blood culture
directly, 1 ml was used in WBC count and the other 2 ml were centrifuged and the serum used for serological tests.
3.2.3 Laboratory Identification of Typhoid Fever 3.2.3.1 Serological Identification (Widal test) - The slide test according to manufacturer, two ml of blood were centrifuged for 5 minutes (5000 rpm) and the serum (upper supernatant) was used for testing. One drop of undiluted serum was put on a microscopic slide, a drop of undiluted antigen suspension was poured on the serum, mixed well, when agglutination seen within three minutes the reaction scored as positive. - Tube test according to manufacturer, all positive results obtained through slide tests were confirmed using the tube agglutination test as follows: 1. Eight test tubes in a rack were labeled up. 2. Used pipette, 1.9 ml of 0.90% normal saline was dispensed into the first tube, and 1.0ml into the remaining seven tubes. 3. Used a pipette 0.1 ml of the patient's undiluted serum was dispensed into the first tube and mixed well. 4. Using a pipette, 1 ml from the first tube was dispensed into the second tube and continued this method to the seven tubes and all were mixed well. The eight tube was containing only saline as a control. 5. 1drop of the appropriate antigen suspension was added into each tube and mixed well.
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Tubes were incubated at 50˚C for 4 hours to Salmonella O antigen and 50˚C for 2 hours to Salmonella H antigen. The titer taken was the last tube showed agglutination. For negative results, all tests remained clear of any agglutination. Tubes were left overnight in a fridge, then allowed to reach room temperature before reading.
3.2.3.2 White blood cell count Prepared according to Heiserman (2004) as follows: 1. Blood was drawn to the 0.5 mark (50Ml) in a white blood cell diluting pipet and diluted to the 11 mark with WBC diluting fluid (2% glacial acetic acid). The blood was washed into the bulb of the pipet, which has a volume of 10(100Ml). 2. The contents of the pipet were mixed for 3-5 minutes to ensure even distribution of cells. 3. The pipet tip was touched to the junction of the cover glass and the counting chamber; the mixture was allowed to flow under the cover glass until the chamber was completely charged, and filled with the opposite chamber of the hemacytometer. 4. The cells were allowed to settle for about 3 minutes, under lowpower
magnification and reduced light, focused on the ruled area
and observed for even distribution of cells. 5. The white cells were counted in the four 1 sq mm corner areas. 6.
The number of WBCs per cubic mm was calculated as follows:
WBCs= Average number of chambers (2) WBCs counted x dilution (20) Volume (0.4)
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3.2.3.3 Isolation of Salmonella enterica Serovar Typhi Biphasic blood culture (Castaneda's method) (Chopra 1985 and Garrity et al., 2004), was used to isolate Salmonella enterica serovar Typhi. Two ml of blood was used to inoculate one biphasic medium bottle, culture was incubated at 37˚C for 24 hours, then subcultured on 7% blood agar, MacConkey agar and SS agar, Negative results continued incubation for seven days and subcultured every 24 hours. Subcultures were as follows: from each blood culture bottle, a loopfull was transferred to blood agar and streaked, incubated for 24 hours at 37˚C, these colonies are non hemolytic, and transferred by a wire loop to a MacConkey′s agar, streaked, incubated for 24 hours at 37˚C, those colonies were non lactose fermenters and produce pale colony (Pollack, 2003 and Chopra 1985), tested for oxidase negative isolates were transferred by a wire loop to SS agar, streaked and incubated for 24 hours at 37˚C. All those colonies appeared colorless with a black center, circular, convex and smooth (Forbes, 2007), are suggested to be Salmonellae, were subjected to biochemical tests and confirmative identification by RapID one system and PCR. Figure (3-1) was put depending on Issak, 2005; Pang et al., 1998; Chopra 1985; Garrity et al., 2004 and Forbes, 2007, as a schematic diagram to identify the isolates grown on biphasic blood media.
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Blood sample ↓ Biphasic culture
Blood agar
MacConkey′s agar
SS agar
Hemolysis Lactose fermenter
-ve
Lactose non fermenter
+ve
-ve +ve Oxidase
+ve
RapID One System
-ve(enterobactericeae)
Kligler Iron agar (Alka/Acid, H2S) Motility test +ve
Figure (3-1) Schematic diagram shows the identification key of Salmonella enterica serovar Typhi and bacteria grown on biphasic blood culture.
3.2.3.4 Gram Stain A small portion of isolated colony was Gram stained on a slide and examined by light microscope (Anija, 2003).
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3.2.3.5 Biochemical test 3.2.3.5.1 Kligler iron agar Kligler iron agar was inoculated by stabbing well isolated colony grown on MacConkey′s agar for 24 hours at 37˚C through the center of the medium to the bottom of the tube and then streaking the surface of the agar slant; the cap was left on loosely and incubated the tubes at 37˚C for 24 hours. K (alkaline)/A (acid) may occur with or without H2S (Black precipitate). Salmonella enterica serovar Typhi is H2S producer.
3.2.3.5.2 Urease test In this test, the surface of Urea agar slant was streaked with a portion of a well isolated colony grown on MacConkey′s agar for 24 hours at 37˚C, and incubated at 37˚C for 24 hours. No color change (agar slant and butt remain orange) is Salmonella enterica serovar Typhi. 3.2.3.5.3 Citrate test Simmons Citrate agar slant was inoculated with a well isolated colony grown on MacConkey′s agar for 24 hours at 37˚C, and incubated at 37˚C for 7 days. Positive result was indicated by color change from green to blue. Only Salmonella enterica serovar Typhi is citrate negative. 3.2.3.5.4 Indol test Peptone water was inoculated with a colony grown on MacConkey′s agar for 24 hours at 37˚C, and incubated at 37˚C for 48 hours, then 0.5ml of Kovacs reagent was added. Positive result was indicated by formation of pink to wine colored ring after the addition of appropriate reagent .Salmonella is non indole former that shows no colored change after adding reagent. 3.2.3.5.5 Methyl Red/Voges-Proskauer (MRVP) Tests Inoculated MRVP broth with a well isolated colony was grown on MacConkey′s agar for 24 hours at 37˚C, and incubated at 37˚C for 48
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hours. For Methyl Red test, 5 drops of methyl red reagent was added for 5 ml of broth. The reaction was read immediately, Salmonella enterica serovar Typhi is methyl red positive through appearance of red color. For Voges- Proskauer (VP), 6 drops were added of solution A (Alfanaphthol) and 2 drops of solution B (KOH) were added to 1ml of MRVP broth, shaked well and observed after 5 minutes. Red color indicates positive result; Salmonella enterica serovar Typhi is VP negative indicated by remaining the color yellow.
3.2.3.5.6 Motility test SemiSolid Agar was inoculated by stabbing to a depth 1/3 to1/2 inch in the middle of the tube with a colony grown on MacConkey′s agar for 24 hours at 37˚C, and incubated in 37˚C for 24 hours. Motile bacteria appeared by spreading out into the medium from the site of inoculation.
3.2.3.5.7 Oxidase Test A single colony grown on MacConkey′s agar for 24 hours at 37˚C was picked by a wooden stick and rubbed on filter paper moisted with the substrate (1% tetramethyl-p-phenylenediamine dihydrochloride), the color changed to deep blue or purple within ten seconds indicates positive result. Salmonella enterica serovar Typhi oxidase negative.
3.2.3.6 Identification of Salmonella enterica serovar Typhi by Remel RapID One System As mentioned in section (3.1.10), 18 reaction cavities of RapID One Panel were inoculated with pure culture grown on MacConkey′s agar for 24 hours at 37˚C, incubated at 37˚C for 4 hours, then the reagent of RapID One were added and allowed at least for 10 seconds for color development. Test of cavities were read and recorded in appropriate box
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of report form, by referencing the microcode for each test on the report form. Identification was recorded according to RapID One Code Compendium.
3.2.4 Storage of bacterial isolates For short term storage, the isolates were maintained by streaking them on surface of Nutrient agar Slants and incubated at 37˚C for 24 hours, then stored at 4˚C for one month. Long terming storage by inoculated Brain heart infusion broth with pure colony and mixing well with glycerol (20%), stored at -20˚C for 6 months (Anija, 2003; Garrity et al., 2004 and Forbes, 2007) .
3.2.5 Genomic DNA Extraction DNA of Salmonella enterica serovar Typhi was extracted using Genomic DNA Purification Kit (Fermentas, Germany), according to manufactures the extraction was included the following: 1. Amount of 10-20 ml bacterial culture (grown in LB broth for 24 hours at 37˚C) was centrifuged for 10 min at 7500 rpm. 2. The supernatant was discarded, and pellet was resuspended in 200µl of TE buffer in a 1.5 ml microcentrifuge tube. 3. 400µl of lysis solution was added to the supernatant and incubated at 65˚C for 5 minutes. 4. Immediately 600µl of chloroform was added and emulsified gently by inverting 3-5 times and centrifuged the sample at 10.000 rpm for 2 min. 5.
Supernatant,
containing
DNA,
was
transferred
to
a
new
microcentrifuge tube; 800µl of fresh prepared precipitation solution was added, mixed gently by several inversions at room temperature for 1-2 min and centrifuged at10.000 rpm for 2 min.
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6. Supernatant was removed completely, and the DNA pellet was dissolved in 100µl of 1.2 M NaCl solution by gentle vortexing. 7. 300µl of cold ethanol was added, DNA was let to precipitate for 10 min at -20˚C and then spinner down for 10.000 rpm at 3-4 min. Ethanol was poured off and DNA was seen visually by electrophoresis in agarose gel. The DNA extracted was used as template DNA for PCR.
3.2.6 Polymerase Chain Reaction DNA amplification was carried out according to Fermentase manual in a total volume 25µl consisting PCR Master Mix which contained:
Volume
Reagents
16.75 µl
DNase free water
2.5 µl
10X Taq buffer (KCl)
0.5µl
dNTPs mix
0.5µl 0.5µl
Forward primer Reverse Primer
2µl
Template DNA
2 µl
MgCl2
0.25µl
Taq DNA polymerase
Total volume 25µl
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The mixture was mixed well in a sterile 1.5 PCR tubes. All PCR tubes (1.5ml) were placed in the Thermal Cycler. For all pairs of primers using the PCR amplification was optimized as follows: (Haque et al., 1999; Woods, 2008; Pathmanathan et al., 2003; Levy et al., 2008 and Bailey et al., 1992).
Cycling conditions Primers
Initial
Denaturation
Annealing
Extension
denaturation
Final
No.
extension
of cycles
hilA for
94˚C for 5
94˚C for 1
65˚C
72˚C
72˚C for
30
hilA rev
min
min
for1min
for1min
10min
cycles
ConOri-F
96˚C for 5
94˚C for 30s
52˚C for
72˚C for
72˚C for
29
ConOri-R
min
30s
1 min
10min
cycles
tyv-s
95˚C for 2
55˚C for
72˚C for
72˚C for
35
tyv-as
min
30s
30s
5min
cycles
ST1 ST3
94˚C for
94˚C for 1
48˚C for
72˚C for
72˚C for
30
ST2 ST4
1min
min
1min
1 min
7min
cycles
95˚C for 30s
The two pairs of primers ST1, ST2 and ST3, ST4 were used in nested PCR amplification by the same methods of other primers instead that the ST1, ST2 amplification product was diluted as 1:5 DNase free water and used as template for ST3, ST4 primers.
3.2.7 Agarose Gel Electrophoresis (Knight and Monroe, 1999 and Biotechnology Index, 2000). All reaction mixture was fractionated electrophoretically in 1% agarose gel contained 0.5µg per ml Ethidium bromide, then photographed
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used a UV transsilluminator and digital camera (Sony, Japan). A master mix without template was used as negative control. Gel preparation and electrophoresis were carried out as follows: 1. One hundred solution of 1% agarose was prepared in TBE buffer. 2. Solution was placed in a microwave oven and heated until agarose was completely melted. 3. Agarose was allowed to cool for 10 min at room temperature, then Ethidium bromide added, all solution was poured into the casting tray, a 16 pin comb was fixed while the gel leaved to cool and solidified for 30 min. 4. After the gel was cooled, the comb was removed; 1µl loading dye was added to 6 µl PCR products, the mixture was pipette into each of the wells of the gel. 5. TBE buffer was poured in the electrophoretic gel tank until became 1-2 mm deep over the gel. 6. Power supply was connected to the Gel Tank, and electrophoresis was run set at 90 volts for 1 hour. 7. The gel was removed and placed on the UV Viewing Cabinet, then the transsilluminator was turned on and photographed.
3.2.8 Statistical analysis The means of typhoid fever tested for independence between different sex, age group, residency and months using Chi- Square (X2) test for independence. The statigraphics program (Statigraphics Corp, 1999) was used in statistical analysis of Chi- Square (X2) test.
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Results and Discussions 4.1 The incidence rate of Typhoid and Paratyphoid fever Initially, to find the feasibility of working on typhoid fever in Kalar, the incidence rate of the disease was calculated for three years, in the period 2005-2007. The incidence rate for both sexes for the three years (appendix, I and Figure, 4-1) was low (0.8, 0.57 and 0.52 respectively) in comparison to findings in near and far countries like India, South Africa, Hong Kong and Egypt; in different parts of India, the morbidity due to typhoid fever in 2006 varied from 102 to 2219 per 105 population, and in some areas, typhoid fever was responsible for 2 to 5% of all deaths (Nagarajan et al., 2009). Data released by the Department of Health showed that the incidence of typhoid fever in South Africa was in the order of 1.04 cases per 105 population per year (Health trends in South Africa) The incidence rate data revealed that typhoid fever is endemic in Kalar but with low value when it compared with a developed country like Hong Kong where typhoid is endemic also with an incidence rate averaged yearly 2.7 cases per 105 populations (Ling et al., 2000) and with Egypt that appeared of high incidence, 10-100/ 105/ year (Fadeel et al., 2004). It was clear that the incidence
rate
for
females
is
significantly
higher
than
males
(P<0.28)*especially in 2005 (Appendix I). This result also confirms the endemic condition of the disease while the interruption of the results for males and females with respect to incidence rates indicates that the opportunity to infection is depending on the chance of exposure to causative agent rather that sex specificity. When the incidence rate was calculated according to age groups, the group 15-60 years was significantly with high incidence rate in comparison to the lowest rate for <5 years group (p< 0.5)** ( appendix, II and Figure, 4-2). *Х2 (2) 0.05=5.99 , **Х2 (3) 0.05=7.8.
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In c id e n c e ra te p e r 1 0 0 .0 0 0 p e rs o n s
1.4 1.2
1.17
1 0.81
0.8
0.8
2005 2006
0.67 0.57
0.6 0.47
0.48
0.44
0.52
2007
0.4 0.2 0 Female
Male
Total
Figure (4-1) Total and sex specific incidence rate of typhoid fever in Kalar district per 105 persons per Year.
It was reported previously that all age groups are susceptible to Salmonella, but the elderly, infants, and persons with impaired immune systems are at increased risk for serious illnesses (Lee, 2002) and has been suggested that since
young adults have a more adventurous lifestyle, they
are therefore at higher risk for food borne diseases (Ekdhal et al., 2005). Higher incidence was also reported for pregnant women (Hasbun et al., 2006). Mortality rate was 0.45 (0.6 for males and 0.36 for females), while in 2005 and 2006 was zero, this means mortality is rare by typhoid fever because of effective treatment. In rural areas, the incidence rate of infection was significantly high in comparison to urban areas (p< 2.7)* (appendix, III and Figure, 4-3). *Х2 (2) 0.05=5.99
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In rural areas, the hygienic awareness is lower than urban in the district
In c id e n c e ra te p e r 1 0 0 .0 0 0 p e rs o n s
under study.
1.4 1.2 1.2 1 0.8
0.7
2005 2006
0.6
0.5 0.5
0.5 0.42
2007
0.4 0.2
0.22
0.19
0.13 0.080.04
0.1
0 <5
6-
15-
>61
Age group (year) Figure (4-2) Age group year’s specific incidence rate of typhoid fever in Kalar district per 105 persons per Year.
However, Salmonella are difficult to eradicate from the environment (Lee, 2002 and Davidson, 2003) and the incidence of Typhoid fever decreases when the level of development of a country increases (Toder, 2005). Most typhoid infection occurs in less industrialized countries where sanitary condition remains poor and water supply is untreated, whilst in industrialized countries two major changes occurred in the pattern of disease, one was marked decline in the total incidence in the past-century;
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the other was that the disease became predominantly associated with travel (Tulchinsky et al., 2000; Guzman et al., 2006 and Kelly et al., 2008). 8
Inciden ce rate p er 100.000 p erson s
7
6.68
6
5 2005 4
2006 2007
3 1.79
2
1 0.018
0.05 0.079 0.079
Rural area
Urban area
0
Figure (4-3) Incidence rate of typhoid fever according to residency of the patients.
So, the incidence either in rural areas or urban areas is related the degree of sanitation which in turn related to the degree of development in spite of that the incidence rate of typhoid in kalar is low that reflects the good hygienic awareness in the district. When the incidence rate was calculated along months of the year, disease appeared but it was significantly upraised in summer season (Jun and July) (p< 0.3)*(appendix, IVand figure, 4-5). *Х2 (22) 0.05=33.9
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Hot and dry seasons favor the disease, while cold and wet seasons tend to check it (Thomas, 1907).Typhoid bacilli are commonly found in water, ice, food, milk, and soil. They do not multiply in water but can survive in ice and ice creams for up to a month and up to 70 days may account for high incidence of enteric infection in summer and it may be able to grow in heavily polluted water in warmer months (Chopra 1985; Lee, 2002; Davidson, 2003; Tulip Group, 2004 and Ekdhal et al., 2005). In well water, these organisms may survive for about 7 days and in sewage polluted water may survive for more than 4 weeks (Chopra 1985). An estimated survival time of Salmonella in the environment is about 3.25 months in the summer season and 3.98 months in the winter season (Lee, 2002). Many infectious diseases including typhoid fever are influenced by climate; climate plays an important role in the transmission process and can influence spatial and seasonal distributions (Burke et al., 2001). Different pathogens, as well as humans, respond to seasonal changes in the environment and that some conditions are more favorable than others for disease transmissions (Kelly et al., 2008). Paratyphoid fever was not reported in 2005, but in 2006 only two cases were reported, they were inpatients while cases increased in 2007 (12 cases), so, the incidence rate was significantly higher in 2007 (p<0.005) *( Figure, 4-4 and appendix, V), increase in disease incidence, either typhoid or paratyphoid, within years following 2003 when the political situation leads to rearrange of people in Iraq according to their original residency may be due to the large number of reimmigrants returned to Kalar from south of Iraq and from Iran as well. The statistical reports showed returning of 1907 families from middle and south of Iraq and 126 persons from Iran to Kalar in 2005 while 9792 families from southern Iraq and 665 persons from Iran were reimmigrated in 2006 ( Non published data of Kalar statistics directory) . *Х2 (1) 0.05=3.8
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In fact, reports considered migration as an important factor in typhoid outbreaks (Provost et al., 2006). Pollack (2003) and NaTHNaC (2007) reported that although over 60% of typhoid and paratyphoid cases are associated with travel, the travel history information is not always available and the number of travel-associated cases is likely to be higher.
0.012
0.01 Incidence rate per 100.000 persons
0.01
0.008 0.007
0.007 2006
0.006
2007
0.004 0.003
0.003
0.002 0 0 -------
-------
------
Female
Male
Total
Figure (4-4) Total and sex specific incidence rate per 105 persons-Year time paratyphoid fever in Kalar region.
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0
0.05
0.1
0.15
0.2
0.25
Jan
Feb
Mar
Apr
May
2005
Jul Months
Jun
2006
Aug
2007
Sep
Oct
Nov
Figure (4-5) Average monthly typhoid fever incidence rate per 105 populations in Kalar for the period 2005-2007.
In c id e n c e ra te % p e r 1 0 0 .0 0 0 p e rs o n s
Dec
4.2 Widal test results Out of 120 clinically suspected typhoid patients examined for widal test in the period between September 2008 to May 2009, 70 (58.3%) were positive according to titers interpreted as positive (1: 160 or more for anti O and anti H antigen) as mentioned by Al-roubaeay (2008) While all 15 healthy control samples were negative, but 13 of them (86.6%) were with 1:80 titer for anti H antigen, table (4-1).
Table (4-1) Widal test results represented as the number and percentage of titer positive patients against O and H antigens of Salmonella enterica serovar Typhi among 120 clinically diagnosed typhoid patients and 15 healthy control persons.
Number and percentage of positive titers Titer
Suspected patients Anti O antigen
Control
Anti H antigen
Anti O antigen
Anti H antigen
No.
%
No.
%
No.
%
No.
%
<1:80
43
35.8
35
29.1
0
0
2
13.3%
1:80
20
16.6
13
10.8
0
0
13
86.6%
1:160
25
20.8
29
24.1
1:320
29
24.1
40
33.3
1:640
3
2.5
3
2.5
Total
120
100
120
100
Total for both
70 (58.3%) *
antigens *Total positive for both antigens includes the titer 1:80 for anti O antigen and 1:160 for anti H antigen and more.
Out of the 70 patients, 19 (15.8%) were males and appendix; VII (figure, 4-6).
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51 (42.5%) females,
Percentage % of positive widal test 70.00% 58.30%
60.00% 50.00%
42.50%
40.00%
Percentage % of positive widal test
30.00% 15.80%
20.00% 10.00% 0.00% Female
Male
Total
Figure (4-6) Percentage of widal test positive patients according to sex group.
The titers showed incompatibility with results of Al-Roubaeay et al. (2008) in Ibn-AL-Khateeb hospital- Baghdad where positive O titers 68 (30%) were higher than 40 (20%) H titers. Also, the results mentioned in Baghdad were similar to reports achieved in Turkey and India where O titers positive were higher than H titers (Willke et al., 2002 and Ambati et al., 2007). Because of that, the higher anti H antigen titer means past vaccination or past infection (Pokhrel et al., 2009), the earliest serological response in acute typhoid fever arisen in the titer of the O antibody, with an elevation of the H-antibody titer developing more slowly but persisting longer (Parry et al.,1999). While for those patients under test who were diagnosed clinically as suspected patients, the number and percentage of reinfection; patients with symptoms reappeared after curing by treatment, were confirmed through the case history of the patients, the results showed high percent of reinfected patients that confirm the higher percent of anti H
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antigen but also results showed that females were with high opportune (figure, 4-7) so as to infect (appendix, VIII). Percentage of reinfected patients 70.00% 62.60%
Percentage of reinfected patients
60.00%
56.60%
50.00% 43.20% 40.00% Percentage of reinfected patients 30.00%
20.00%
10.00%
0.00% Female
Male
Total
Figure (4-7) Reinfections in male and female in suspected cases of typhoid fever.
For the elevated titer of anti H antigen for 13 out of the 15 healthy persons (86.6%) which at once pointed to past infections by Salmonella enterica serotype Typhi according to Pokhrel et al. (2009), the results showed some interruption that out of 120 suspected patients, 10.8% were with titer 1:80, this means that titer 1:80 is with higher ratio in control samples than suspected patients, so if it was kept in mind that the H antigen is polyclonal, Commercial antiflagellar antibodies could contain antibodies against the O antigens from the immunizing organisms, which could explain the cross reactivity (Rementeria, et al., 2009), also would be expected from the antigenic pattern of the indigenous salmonellae may be natural antibodies unrelated to antigenic stimulation (Oyeyinka and
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Salimonu, 2002). It is not satisfactory to see high ratio of 1:80 for anti H antigen titer among healthy persons, so that the 1:80 titer for anti H antigen should not be depended on diagnosis of typhoid fever. According to Parry et al. (1999), some authors have claimed that the level of H agglutinins is unhelpful in the diagnosis of typhoid, maintaining that the H-agglutinin titer remains elevated for a longer period than the O-agglutinin titer after an episode of typhoid fever and also may rise as a nonspecific response to other infections. It is not known why women are more commonly carriers than men (Chopra, 1985), and nor why the disease ratio for typhoid and paratyphoid was higher for females compared with the other enteric diseases (Mitchel et al, 2002), but Khan (2004) reviewed the severity of typhoid fever in general and hepatic dysfunction in particular which was most marked in adult women, 98.5% (64/65) of whom were in the reproductive age group (15-45 years). He mentioned that Estrogens have various stimulatory effects on macrophages, including Kupffer cells that being considered primary sites of localization of S. typhi within the liver. The number of cases with suspected typhoid fever and positive for widal test were 70, from these cases 43 out of 120 cases tested (35.8%) were mixed infections (typhoid and paratyphoid fever) by positive results for widal test (Salmonella enterica serovar Typhi antigens O, H and Salmonella enterica serovar Paratyphi B antigen O and H) (Table, 4-2). This means that widal test is not to rely as significant test for diagnosis of typhoid and paratyphoid fever. Major problems relate to the difficulty of interpreting Widal test results in areas where Salmonella typhi is endemic and where the antibody titers of the normal population are often not known. Furthermore, in areas where fever due to infectious causes is a common occurrence, the possibility exists that false-positive reaction may occur as a result of non-typhoid fevers (Pang and Puthucheary, 1983).
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Table (4-2) Widal test results represented as the number and percentage against O and H antigens of Salmonella enterica serovar Paratyphi B in 120 clinically diagnosed typhoid patients.
Number and percentage of positive patients Titer
O Antigens
H Antigens
No.
%
No.
%
≥1:40
66
55
68
56.6
1:80
10
8.3
17
14.1
1:160
25
20.8
15
12.5
1:320
18
15
20
16.6
1:640
1
0.8
0
0
Total
120
100%
120
100%
While Salmonella enterica serovar Paratyphi C and Salmonella serovar Paratyphi A were negative for widal test compatible to results of Athawra General Hospital, Baghdad (Maikan,2006), but in Ibn-ALKhateeb hospital, Baghdad, Salmonella paratyphi A was 0.78% in 2008 (Al-Roubaeay et al., 2008) and in Basra 3.33%
(AL-Tammimi, 2001). The distribution of
paratyphoid bacilli shows marked geographical differences. Salmonella paratyphi A is prevalent in India and other Asian countries, Eastern Europe and South America (Chopra, 1985; Tankhiwale et al., 2003 and Ochiai et al., 2005), and the proportion of Salmonella paratyphi infection would have shown an increase in non immunized as well as immunized travelers, typhoid vaccination may cause a shift toward a higher percentage of Salmonella paratyphi A infection since travelers are not protected against it (Meltzer et al., 2005). Salmonella paratyphi B is prevalent in Western Europe, Britain and N. America, and Salmonella paratyphi C in Eastern Europe and Guyana (Chopra, 1985). Possible cause of high infection with
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paratyphoid organisms in Europe is that they use prepared food products more than people of underdeveloped countries and paratyphoid is mostly spread by meat, fish and other prepared foods (Ahmed et al., 2007). In Kalar, most people do not use prepared food products which may therefore be the reason of low infections by paratyphoid fever. In any cases, Salmonella paratyphi C infections are rare and usually sporadic (Chopra, 1985; Freidin, et al.1985). For practical purpose, the complete blood count in enteric fever is unremarkable (Kundu et al., 2006), the WBC count is normal in most cases and leukocytosis makes the diagnosis less probable, Leukopenia perceived to be an important feature of typhoid fever and has been reported in only 20-25% (Abdool Gafar et al., 1992) cases and according to Sharma et al. (2003), 32% have low leucocyte count. In the current study, also appeared that the white blood count was within normal value for all 120 patients where the range was 4- 6×109/1for all the 70 widal test positive patients (appendix, IX). According to findings of Abdool Gafar et al., (1992) and Parry et al. (2002), the current study result confirms unremarkability of WBC count dependent diagnosis of typhoid or paratyphoid fever. The clinical diagnosis of typhoid fever is difficult and its definitive diagnosis requires the isolation of Salmonella typhi from the patient (Willke et al., 2002; Mohanty and Ramana 2007). Levine et al. (1978) and Nagarajan, (2009) mentioned that Widal result may be less sensitivity and less specificity, particularly in a community with endemic typhoid fever. Widal test also depends on the level of infection due to other salmonellae with cross-reacting antigens that cross-reactions lead to produce false-positive O-antigen titer in the Widal test (Parry et al., 1999 and Vandepitte et al., 2003).
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4.3 Isolation and primary Identification of Salmonellae Out of 120 suspected typhoid patients, 70 of them were positive for blood culture by using biphasic blood medium. Subculturing on blood agar, MacConkey′s agar and SS agar followed by further biochemical and physiological tests revealed isolation of primary (14) identified Salmonella enterica serovar Typhi isolates, while those isolates which were non Salmonellae were tested for further identification according to the scheme shown in figure (3-1), which led to isolate Escherichia coli (11 isolates) (9.1%), and 7 (5.8%) Brucella isolates while 38 (31.6%) remained unidentified. (Figure, 4-8). All the biphasic culture positive persons were the same positive for widal test. All (15) control persons showed no growth on biphasic blood culture. All Salmonella enterica serovar Typhi isolated were gram negative coccobacilli shaped, non encapsulated, non spore forming, negative for urease, Voges Proskauer and oxidase test, positive for Methyl Red and motility, while in kligler test they produced H2S with acid bottom and glucose fermenter and variable results for Simmon citrate agar. Brucella isolates were oxidase positive, non lactose and non glucose fermenters on kligler Iron agar, gram negative bacilli, urease positive, negative for Methyl Red, Voges Proskauer, Simmon citrate agar, only one of the Brucella isolates were H2S producers (P105). Only (2) isolates, out of the 38 unidentified isolates, were gram positive coccal, non spore forming and lactose fermenters. Several diseases caused by non-Salmonella organisms, like brucellosis, have been shown to exhibit cross-reactivity in typhoid endemic regions, and these cross-reactions increase the error rate of the result of the Widal test (Adams, 1978; Thaveri et al., 1995 and Willke et al., 2002). The isolation percentage of Salmonella enterica serovar Typhi was 11.6%, figure 4-8 out of the 120 suspected typhoid patients tested. This percent was higher than that obtained by AlRoubaeay(2008) in Baghdad (8.23%) who used blood culture for primary
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isolation( but not biphasic blood culture) and lower than
the percent
obtained by Al- Tammimi (2001) in Basra (19.44%) who used Api 20E system for identification. However typhoid fever is seemed to be endemic in Iraq.
Percent of bacterial isolates
35.00%
31.60%
30.00% 25.00% 20.00% % isolates
15.00%
11.60% 9.16%
10.00%
5.80% 5.00%
ct
er ia
ce lla U
ni de
nt i fi
ed
ba
Br u
E. co li
Sa lm
on
el la
0.00%
Figure (4-8): the percentage of bacterial isolates isolated from blood samples of 120 suspected typhoid patients identified primarily.
The Salmonella isolates were further tested by RapID One system (section, 4-4) and PCR (section 4-5) for complete identification. It was appeared that not all Widal test positive patients were positive for Salmonella isolation. Salmonella enterica serovar Typhi shares these antigens with other diseases caused by non-Salmonella organisms (malaria, dengue,
miliary
tuberculosis,
endocarditis,
chronic
liver
disease,
brucellosis, etc), other Salmonella serotypes and shares cross-reacting antibodies from infections with other gram-negative enteric bacilli. Furthermore, acute typhoid cases may mount no detectable antibody
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response or have no demonstrable rise in antibody titer (Parry et al., 2002; Olopoenia and King, 2000 and Adams, 1978). The positive results of Widal test for those Salmonella negative isolation persons, in spite of that, it may be due to false positive reactions or past infection as mentioned earlier, the problem may be latent in the isolation of Salmonella by blood culture. Pang & Puthucheary (1983) and Haque et al. (1999) pointed to some factors affect in the isolation of Salmonella from blood such as the very few numbers of bacteria needed to cause sever infection, bacteriostatic effect of antibiotics administered before the culture sample is taken, the host’s immune response system, and the intracellular characteristics of Salmonella typhi. In fact, for perfect isolation of Salmonella from blood, Sodium polyanethol sulfonate (SPS) should be added to medium for its anticomplementary and antiphagocytic effect (Forbes et al., 2007), but unfortunately this substance was not available in local markets to be used. Except one inpatient who was on antibiotics, all others (119) were outpatients and the history of antibiotic administration was depending on the answers of patients when they were asked (Appendix,XII).
4.4 Identification of salmonella using RapID One System The (14) Salmonella enterica serovar Typhi isolates identified primarily by blood culture and biochemical tests as well as the (11) isolates of E. coli which identified by the same way were tested by RapID One system to confirm identification. All Salmonellae isolates had the same reactions, also E. coli (Table, 4-3). So, this result confirmed that the identification scheme used (section 3.2.3.3, figure, 3-1) is reliable and with valid results, but the use of RapID One system in addition to this scheme is required to give confirmative results. However, RapID One system is with high sensitivity, easy to use with rapid results and with low cost.
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Table (4-3): RapID One System for (14) isolates of primarily identified Salmonella enterica serovar Typhi (all with same reactions) and (11) isolates of Escherichia coli (all isolates of the same species are with same reactions).
Cavity
Test Code
1
Results* Salmonella enterica serovar Typhi
Escherichia coli
URE
Yellow
Yellow
2
ADH
Yellow
Yellow
3
ODC
Yellow
Blue
4
LDC
Blue
Yellow
5
TET
Orange
Orange
6
LIP
Orange
Orange
7
KSF
Orange
Orange
8
SBL
Yellow
Yellow
9
GUR
colorless
Yellow
10
ONPG
colorless
Yellow
11
BGLU
colorless
Colorless
12
BXYL
colorless
Colorless
13
NAG
colorless
Colorless
14
MAL
Yellow
Yellow
15
PRO
colorless
Colorless
16
GGT
Violet
Colorless
17
PYR
colorless
Colorless
18
ADON
Red
Red
18
IND
Red
Brown
*Code for Salmonella enterica srovar Typhi (0120010) and for Escherichia coli (4061001)
The accuracy rate of RapID One system was reported to exceed 91%, (Hara, 2005; Dottie, 2004 and Dipersio et al., 1984), so that the RapID One system is more reliable and is advised to use it in the identification of Salmonella serovars that in this study the serovar was determined by this system even if the serotyping for isolates was not applied. However,
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identification by RapID One system needs the primary isolation of bacteria on biphasic blood culture and obtaining a pure culture.
4.5 Identification by PCR for Salmonella isolates During this work, the preserved cultures of 8 Salmonella enterica serovar Typhi, out of 14 isolates, were contaminated and as a result they were lost, so only six (P3, P19, P41, P68, P76, and P81) (Appendix XII) were remained and tested by polymerase chain reaction PCR. When the universal primers for identification of Salmonella genus members were used, all six isolates showed amplification by using the primers ConOri F and ConOri R (figure, 4-9). The primers ConoriF/ Conori R are specific for the origin of replication sequence (OriC) which is a genus specific locus, the bands were as expected, appeared of 461bp for each which detect Salmonella strains (Woods, 2008). Out of six isolates tested for Salmonella genus members, only four (P3, P41, P68, and P81) showed positive results for amplification by using the primers US/DS (figure, 4-10), the four isolates appeared to be of 784bp (Guo et al. 2000; Castro et al. 2002; Churi 2004 and Jimenez &Castro 2004 and Pathmanathan et al. 2003) which confirm the Salmonella members while the two non amplified isolates (P19 and P76) were seemed to be hilA and then Salmonella Pathogenicity Island 1 (SPI-1) deficient.
More than 25 genes needed to encode SPI-1 of
Salmonella invasion gene located at centisome 63 (Gelan 1996 and Darwin and Miller 1999), hilA (hyperinvasive locus A), is one of them and it is required for the regulation of the type III secretion genes. The bacteria initiate invasion by expressing a type III secretion system that mediate injection of effector proteins from the bacteria into the cytoplasm of the eukaryotic cell. These genes are required for the initial invasion of the intestinal epithelium that is essential for Salmonella to cause localized
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gastroenteritis, as well as systemic disease (Lostroh et al., 2000; Mirold et al., 2001; Darwin &Miller1999 and Haraga et al., 2008), they are present in all invasive strains of Salmonella and absent from closely related genera such as Escherichia. (Pathmanathan et al., 2003; Jimenez and Castro, 2004).
M
I
II
III
IV
V
VI
900 800 700 600 500 400 300
461bp↑
200 100
Figure (4-9): Agarose gel electrophoresis of PCR products for DNA amplification using the ConOriF/ConOriR primers for six identified primarily by RapID One system M: 100bp plus DNA ladder marker. Lane I: P81, Lane II: P76, Lane III: P3, Lane IV: P68, Lane V: P41, Lane VI: P19.
hilA were used as a molecular diagnosis tool of Salmonella enterica serovar Typhi to detect pathogenicity by SPI-1 while the pathogenicity predicated on the absences of SPI-1 in human disease-causing strains of
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Salmonella enterica serovar Typhi has been shown to occur in a manner dependent on SPI-2 as mentioned by Hu et al. (2008) regarding that SPI-1 was considered to be essential for intestinal Salmonella pathogenicity (Coombes et al., 2005 and Hapfelmeier et al., 2005).
M
I
II
III
IV
V
VI
900 800→ 700→ 600→
↑784bp
500→ 400→ 300→ 200→ 100→
Figure (4-10): Agarose gel electrophoresis of PCR products for DNA amplification using the US/DS primers for six Salmonella enterica serovar Typhi identified primarily by RapID One system. M: 100bp plus DNA ladder marker, Lane I: P76, Lane II: P19, Lane III: P68, Lane IV: P3, Lane V: P41 and Lane VI: P81.
When the specific primers for Salmonella enterica serovar Typhi were used, ST1/ST2 for Salmonella typhi and Salmonella muenchen and (tyv-s/ tyv-as) for Salmonella O9 group, O factor specific repeating units is in the
di-deoxyhexose
branch unit attached to the mannose which is
tyvelose in 9, 12 (Murray, et al., 1984), five isolates, out of six, (P3, P76, P41, P19 and P81) showed amplification for the two primer pairs (Figure, 4-11 and figure, 4-12). The primers (ST1/ST2) reported by Song et al. (1993) and modified by Frankel, (1994) which target the flagellin gene of
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Salmonella typhi, they were used for regular PCR to amplify a 495 bp fragment corresponding to nucleotides 1036-1056 and 1513-1530 of Hl-d ; the nucleotide sequences and predicted amino acid sequences of region VI, only Salmonella typhi and Salmonella muenchen sequences are amplified whereas 458-bp fragment corresponding to nucleotides 1063–1530 of Hl-d give the same detection (Salmonella typhi and Salmonella muenchen) (Hatta and Smtts, 2007 and Song et al., 1993). So that, one of the six Salmonella isolates (P68) detected by the Universal primers was appeared not to be a member of Salmonella typhi when it is tested by specific primers.
On the application of nested PCR for the five isolate’s PCR
amplification products that amplified by ST1/ST2 primers and confirmed the isolates as Salmonella typhi, all showed amplification by using ST3/ST4 primers (figure, 4-13). ST3 and ST4 were used to amplify a 363 bp fragment of Salmonella typhi corresponding to nucleotide 1072-1089 and 1416-1435 respectively of the flagellin gene, it is preferred to target the flagellin gene because its hyper variable region Vi is unique for Salmonella typhi and provides 100% amplification specificity (Song et al., 1993 and Frankel, 1994). So that, Haque et al. (2001) used the current method of nested PCR to get highly specific results because an alternative method used by hashimoto et al. (1995), in which Vi gene was targeted, can give false positive results because it is also found in Salmonella paratyphi C. The gene tyv encodes CDP-tyvelose epimerase, which converts CDPparatose to CDP-tyvelose (Hirose, 2002; Morine et al., 2004; Banavandi et al. 2005 and Levy et al., 2008). The tyv gene is present in both serovars Typhi and Paratyphi A, but the tyv gene of serovar Paratyphi A does not produce active CDP-tyvelose epimerase due to the 1-bp deletion which causes the frame shift mutation and converts codon 4 of Tyv to a stop codon.
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M 900 800
I
II
III
IV
V
VI
→ →
700 → 600 → 500 → 400 → 300 → 200 → 100 →
↑495bp
Figure (4-11): Agarose gel electrophoresis of PCR products for DNA amplification using the ST1/ST2 primers for six Salmonella enterica serovar Typhi identified primarily by RapID One system. M: 100bp plus DNA ladder marker, Lane I: P76, Lane II: P41, Lane III: P3, Lane IV: P68, Lane V:
P81, Lane VI: P19.
The deleted region was used for the design of primer tyv-s to specifically detect the tyv gene of serovar Typhi but not of serovar Paratyphi A (Verma and Reeves 1989). In general, tyv primers detected isolates of the Salmonella O9 group (Woo et al., 2001 and Hirose et al., 2002).
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M
I
II
III
IV
V
VI
1000→ 900→ 800→ 700→ 600→ 500→ 400→ 300→
↑ 615bp
200→
100→
Figure (4-12): Agarose gel electrophoresis of PCR products for DNA amplification using the tyv-s/ tyv-as primers for six Salmonella enterica serovar Typhi identified primarily by RapID One system. M: 100bp plus DNA ladder marker, Lane I: Lane II: P68, Lane III: P3, Lane IV: P19, Lane V:
P76,
P81, Lane VI: P41.
Table (4-4) showed that all six isolates (P3, P41, P68, P19, P76, P81) were amplified by using Salmonella specific primers (ConOriF/ConOriR) but amplification by using hilA gene primers (US/DS) revealed that four isolates of them were amplified only(P3, P41, P68, P81) whereas the two non amplified isolates P76 and P19 were amplified by using Salmonella enterica serovar Typhi specific primers (ST1/ST2) and (tyv-s/tyv-as). We conclude that (P19 and P76) are Salmonella enterica serovar Typhi but lack hilA gene. hilA and invA genes encoded SPI-1, are considered as molecular markers for the detection of enteropathogenic Salmonella enterica (Malorny et al., 2003 and Arnold et al., 2004) regarding that no previously characterized strains of human enteropathogenicity inducing
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Salmonella lacking SPI-1 have ever been reported (Hu et al., 2008). This result revealed that SPI-1 is essential for enteropathogenicity of Salmonella members, in spite of that enteropathogenicity may occur depending on SPI2 in the absence of SPI-1, but typhoid fever is not dependent on SPI-1, it was appeared clearly to be depended on O9 antigens which are detected in the SPI-1 deficient salmonellae by the tyv-s/tyv-as primers.
M
I
II
III
IV
V
VI
900 800 700 600 500 400→
300 200
↑ 363bp
100
Figure (4-13): Agarose gel electrophoresis of nested PCR products for DNA amplification using the ST3l, ST4 primers for six Salmonella enterica serovar Typhi PCR products amplified by ST1/ST2 primers. M: 100bp plus DNA ladder marker, Lane I: not loaded, Lane II: P19, Lane III: P41, Lane IV: P3, Lane V: P76, Lane VI: P81.
(Table 4-4): The amplification pattern of six Salmonella enterica serovar Typhi by using different primers, primarily identified by blood culture, biochemical tests and RapID one system.
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Primers
Isolates Con OriF/ Con OriR US/DS
ST1/ST2
tyv-s/tyv-as
ST3/ST4*
P19
+
-
+
+
+
P76
+
-
+
+
+
P3
+
+
+
+
+
P41
+
+
+
+
+
P81
+
+
+
+
+
P68
+
+
-
-
NT••
*: Amplification by ST3/ST4 was achieved by nested PCR for ST1/ST2 amplification product. ••: not tested
+: Amplification -: No amplification Salmonella pathogenicity island 1 (SPI-1) is required for efficient invasion of the intestinal epithelium and intestinal disease and SPI-2 is essential for Salmonella replication and survival within macrophages and the progression of a systemic infection , SPI-1 genes have also shown to be important for Salmonella persistence during long-term systemic infections, phagosome maturation, and intracellular proliferation while SPI-2 genes have implicated in intestinal colonization, persistence during the intestinal phase, and induction of secretory and inflammatory responses (Bustamante et al., 2008). In this study, amplification by nested PCR to amplify the ST1 and ST2 products by ST3 and ST4 in addition to using the primers tyv-s and tyv-as was highly specific in detecting S. typhi DNA. Five, out of six samples, were showed amplification in ST1/ST2, ST3/ST4 and tyv-s/ tyvas, this means only one sample (P68) detects no Salmonella anterica serovar Typhi and if it is known that tyv gene is found only in O9 Salmonella whereas S. muenchen is not belonging to O9 group, not all
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Salmonella enterica serovar Typhi identified by interpretation of RapID One system were thus virulent variants (typhoid causatives) but it was appeared that one of the 14 isolates was confirmed microbiologically to be a Salmonella enterica serovar Typhi (P68) and seemed to be another Salmonella variant because the flagellin (Hd-1) and O9 group (tyv) genes were not amplified by ST1/ST2 and tyv-s/ tyv-as primers which are essential for Salmonella enterica to be a Typhi variant in spite of that they were identified as this serovar by cultivation on biphasic blood culture, biochemical tests and RapID One system. This result leads to argument if there are Salmonella enterica serovar Typhi variants cause typhoid but is not depended on tyv, this needs more investigation. The four serotypes of salmonella cause enteric fever (Typhi, Paratyphi A, B and C) can be identified in the clinical laboratory by biochemical tests with API20E (BioMerieux, Marcy1'Etoile) and by serology for O and H antigens (Murex Biotech), the Widal test was performed with Salmonella typhi O and H antigens (Lorne Laboratories) (Issak, 2005). Diagnosis by isolation of Salmonella typhi by culture is a reliable test, so that other tests were developed that one of them was PCR regarding that this test is not yet widely used in routine diagnostics in spite of several studies described the use of PCR in diagnosis of typhoid fever (Pang etal., 1998).
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Conclusions 1. Typhoid fever is endemic in kalar but with low incidence and mortality rate.
2. Females, age group (16-60) years and rural area are more affected. 3. The incidence rate of typhoid fever increases in summer (June and July).
4. Widal test is useful in primary diagnosis but is not reliable. 5. Biphasic blood culture and Remel RapID One System seemed to be accurate methods for Salmonella enterica serovar Typhi identification.
6. Not
all
Salmonella
enterica
serovar
Typhi
have
the
enteropathogenicity encoding hilA gene for this gene is infeasible.
7. Targeting of flagellin and O9 group genes by amplification by regular and nested PCR makes it an excellent tool for diagnosis of typhoid but this needs revision because there was Salmonella identified microbiologically as Salmonella enterica serovar Typhi while it was flagellin gene (Hd-1) and O9 group (tyv) deficient.
8. Brucella was isolated and identified for 5.8% of clinically suspected typhoid patients.
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Recommendations 1. For hilA deficient Salmonella enterica serovar Typhi, that has not been detected by PCR should be investigated if whether is due to a mutation or to completely gene loss because it was reported to be essential for detection of this variant, i.e. a mutant SPI-1 or lost. 2. Investigation of whether the failure in detection of flagellin and O9 group genes in Salmonella isolates that microbiologically identified as Salmonella enterica serovar Typhi means no detection of this variant. 3. Use of Vi antigen in serological identification of carriers in future. 4. Detection of Salmonella enterica serovar Typhi from blood by PCR directly in addition to Brucella when detection procedures applied.
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Arabic references ( ﻋﺰل وﺗﻨﻘﯿﮫ ﻣﺴﺘﻀﺪﯾﻦ ﻧﻮﻋﯿﯿﻦ ﻣﻦ ﻋﺰﻟﮫ ﻣﺤﻠﯿ ﮫ2001) • اﻟﺘﻤﯿﻤﻲ ‚ﺗﻤﺎﺿﺮ ﻣﺤﻤﺪ ﺧﺮﯾﺒﻂ واﺳ ﺘﺨﺪاﻣﮭﻤﺎ ﻓ ﻲ اﺧﺘﺒ ﺎر اﻟﻤﻌ ﺎﯾﯿﺮه اﻻﻣﺘ ﺼﺎﺻﯿﮫ اﻟﻤﻨﺎﻋﯿ ﮫSalmonell typhiﻟﺠﺮﺛﻮﻣﮫ . ﺟﺎﻣﻌﮫ اﻟﺒﺼﺮة. ﻛﻠﯿﮫ اﻟﻌﻠﻮم.( رﺳﺎﻟﮫ ﻣﺎﺟﺴﺘﯿﺮELISA).اﻟﻤﺮﺗﺒﻄﮫ ﺑﺎﻻﻧﺰﯾﻢ
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Appendix (I): Total and sex specific incidence rate of typhoid fever in Kalar district per 105 persons per Year. Year
Female*
Male**
Total†
2005
1.17
0.44
0.8
2006
0.47
0.67
0.57
2007
0.81
0.48
0.52
*: Female per 105females in population. **: Male per105 males in population. †: Total incidence rate per 105 in all population.
Appendix (II): Age group specific incidence rate of typhoid fever in Kalar district per 105 persons per Year.
Year
<5
6-14
15-60
>61
2005
0.08
0.5
1.2
0.1
2006
0.04
0.5
0.5
0.7
2007
0.13
0.19
0.42
0.22
Appendix (III): Incidence rate of typhoid fever according to residency of the patients. Year
Rural area
Urban area
2005
6.68
0.05
2006
0.018
0.079
2007
1.79
0.079
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Appendix
Appendix (IV): Average monthly typhoid fever incidence rate per 105 populations in Kalar for the period 2005-2007.
Year
Jan
Feb Mar Apr May Jun Jul Aug Sep Oct Nov
2005
Dec
0.06
0.1
0.2
0.16 0.11 0.08
0.04
0.01
0.002
0.15 0.08 0.04 0.007
2006
0.02
0.018 0.013 0.018
0.05
0.1
0.02
0.002
2007
0.017
0.006 0.015
0.03
0.09 0.07 0.06 0.05 0.039 0.039
0.038
0.05
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Appendix
Appendix (V): Total and sex specific incidence rate per 105 personsYear time paratyphoid fever in Kalar region. Year
Female
Male
Total
2005
-------
-------
------
2006
-------
0.003
0.003
2007
0.01
0.007
0.007
Appendix (VI): Incidence rate of typhoid fever according to Outpatient and Inpatient. Year
Outpatient
Inpatient
2005
0.79
0.008
2006
0.53
0.0039
2007
1.48
0.0039
Appendix (VII): Number and percentage of widal test positive patients according to sex group. Sex
No.
%
Female
51
42.5
Male
19
15.8
Total
70
58.3
Appendix (VIII):Comparative study of reinfections in male and female in suspected cases of typhoid fever. Sex
No. patients
No.
%
Female
83
52
62.6
Male
37
16
43.2
Total
120
68
56.6
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Appendix
Appendix (IX) :Total White blood cell count in suspected cases of typhoid fever. WBC count
No of patient
Percentage%
4000-11000 / mm3
70
100%
Appendix( X): Results of three different tests; PCR, Blood culture and Widal test for 120 suspected cases of typhoid fever regarding that PCR was done for pure cultures but not for direct detection in blood. tests
No. & percent of cases
PCR
Blood culture
Widal test
5 (4.1%)
+
+
+
1 (0.8%)
-
+
+
50 (41.6%)
-
-
-
56 (46.6%)
-
-
+
8 (6.6%)
NT
+
+
NT: Not tested due to loss during work.
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Appendix
Appendix( XII): Widal test results for 120 patients suspected to be infected by typhoid included in the study. Patient’s serial
Salmonella enterica serovar Typhi
Salmonella enterica serovar Paratyphi B
Antibiotic administration prior testing
Anti O
Anti H
Anti O
Anti H
titers
titers
titers
titers
P1
1/20
1/40
-------------- -------------
- ve
P2
1/40
1/160
--------------- -------------
-ve
P3
1/640
1/320
P4
1/160
1/160
--------------- -------------
-ve
P5
1/40
1/80
-------------- -------------
-ve
P6
--------------
-------------
--------------- -------------
-ve
P7
1/40
1/40
P8
-------------
P9
number
1/320
-------------
1/320
-ve
------------
+ve
--------------
--------------- -------------
-ve
1/40
1/160
--------------- -------------
+ve
P10
-------------
1/20
--------------- -------------
-ve
P11
--------------
-------------
--------------- -------------
-ve
P12
1/160
1/160
-------------- -------------
-ve
P13
1/320
1/320
1/320
P14
1/320
1/320
--------------
P15
----------
-------------
1/160
1/40
-ve
P16
----------
------------
---------------
------------
-ve
P17
1/20
1/20
-------------- -------------
-ve
P18
----------
----------
-------------
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1/320 -----------
1/40
-ve -ve
-ve
Appendix
P19
1/160
1/160
-------------
P20
1/80
1/320
P21
1/20
1/20
-------------
P22
1/20
1/20
------------
P23
-----------
1/40
-------------- -------------
P24
1/320
1/320
P25
1/80
1/80
P26
1/40
1/40
------------ -------------
+ve
P27
1/160
1/160
------------
------------
-ve
P28
1/320
1/320
1/320
1/320
-ve
P29
1/320
1/320
1/160
1/320
-ve
P30
1/80
1/160
1/160
1/320
-ve
P31
1/80
1/160
1/80
1/160
-ve
P32
1/320
1/320
1/160
1/160
-ve
P33
1/80
1/80
-----------
---------
-ve
P34
1/80
1/800
--------
------------
-ve
P35
1/320
1/320
1/320
1/320
-ve
P36
1/160
1/320
1/160
1/80
-ve
P37
1/80
1/40
1/40
1/40
-ve
P38
1/80
1/320
1/320
1/320
-ve
P39
1/320
1/320
---------
---------
-ve
P40
1/320
1/80
1/80
1/320
-ve
P41
1/460
1/320
1/160
1/160
-ve
P42
1/80
1/160
1/80
1/320
-ve
1/320
-----------1/320
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----------
-ve
1/320
-ve
1/20
-ve
-----------
-ve -ve
------------
-ve
1/80
-ve
Appendix
P43
1/40
1/80
------------
1/20
-ve
P44
--------
---------
-----------
---------
-ve
P45
-------------
1/160
-----------
-----------
-ve
P46
----------
-----------
--------------
------------
-ve
P47
1/20
-----------
1/20
----------
-ve
P48
1/320
1/320
1/160
1/80
-ve
P49
1/20
----------
------------
----------
-ve
P50
1/320
1/320
-------------
----------
-ve
P51
----------
1/160
---------
---------
-ve
P52
1/80
1/320
1/80
1/320
-ve
P53
------------
--------------
-----------
----------
-ve
P54
-------------
-------------
-------------
-------------
-ve
P55
1/40
1/40
------------
-----------
-ve
P56
1/40
1/40
---------
-----------
-ve
P57
1/640
1/320
---------
------------
+ve
P58
----------
1/20
-----------
1/20
-ve
P59
---------
-----------
------------
---------
+ve
P60
1/160
1/160
1/80
1/160
-ve
P61
------------
----------
1/20
----------
-ve
P62
1/80
1/160
1/320
1/320
-ve
P63
1/80
1/320
1/160
1/40
-ve
P64
1/160
1/80
1/320
1/80
+ve
P65
1/320
1/320
1/640
1/160
-ve
P66
---------
1/320
1/320
1/160
-ve
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Appendix
P67
1/320
1/320
1/320
1/320
-ve
P68
1/320
1/320
1/320
1/320
-ve
P69
1/80
1/320
1/320
1/320
-ve
P70
1/320
1/640
1/320
1/160
-ve
P71
1/40
1/320
----------
-----------
-ve
P72
1/160
1/160
1/160
1/320
-ve
P73
1/160
1/160
---------
-----------
-ve
P74
1/320
1/320
1/320
1/80
-ve
P75
1/320
1/320
----------
----------
-ve
P76
1/320
1/320
----------
----------
-ve
P77
1/320
1/320
1/160
1/80
-ve
P78
------------
----------
------------
---------
-ve
P79
1/320
1/80
1/320
1/80
-ve
P80
1/80
1/80
--------
----------
-ve
P81
1/320
1/320
--------
-----------
-ve
P82
1/320
1/320
----------
-----------
-ve
P83
1/320
1/640
1/160
1/320
-ve
P84
1/160
1/160
----------
----------
-ve
P85
1/160
1/160
----------
----------
-ve
P86
1/160
1/160
----------
----------
-ve
P87
1/80
1/160
-----------
------------
-ve
P88
1/160
1/160
----------
-------------
-ve
P89
1/20
-----------
------------
----------
-ve
P90
1/160
1/160
----------
-------------
-ve
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Appendix
P91
1/320
1/320
---------
------------
-ve
P92
------------
-----------
-------------
-----------
-ve
P93
1/320
1/320
1/320
1/320
-ve
P94
1/320
1/320
1/160
1/320
-ve
P95
1/160
1/160
1/160
1/160
-ve
P96
1/160
1/160
-------------
------------
-ve
P97
---------
-----------
1/160
1/40
-ve
P98
--------
--------
----------
--------
-ve
P99
-------
-----------
----------
-----------
-ve
P100
------------
1/80
-----------
1/160
-ve
P101
-----------
------------
------------
1/80
-ve
P102
1/320
1/160
------------
-----------
-ve
P103
1/80
1/80
1/80
1/80
-ve
P104
1/80
1/80
1/80
1/80
-ve
P105
1/160
1/160
1/80
1/80
-ve
P106
1/160
1/160
1/160
1/160
-ve
P107
-----------
----------
-----------
1/40
-ve
P108
-----------
1/80
1/160
----------
-ve
P109
1/160
1/320
1/160
1/160
-ve
P110
1/160
1/320
1/160
1/80
-ve
P111
1/160
1/320
1/160
1/160
-ve
P112
1/80
1/320
1/160
1/80
-ve
P113
1/320
1/640
1/320
1/320
-ve
P114
1/80
1/320
1/160
1/80
-ve
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Appendix
P115
1/160
1/160
1/160
1/80
-ve
P116
1/80
1/80
1/80
1/160
-ve
P117
1/160
1/320
1/160
1/80
-ve
P118
1/160
1/160
1/160
1/160
-ve
P119
1/320
1/320
1/160
1/80
-ve
P120
1/160
1/160
1/160
1/160
-ve
P: from patient. +ve: administration of antibiotics. –ve: antibiotics not administrated.
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Appendix
Appendix (XIII): The statistical data of Kalar district residents for the years 2005, 2006 and 2007.
Population Year
Sex
Residency
Age groups
Total
Males
Females
Urban area
Rural area
<5
6-14
15-60
>61
2005
51346
50486
90223
11609
11735
23296
60711
6090
101832
2006
65042
65186
101832
31539
2061
25183
100012
6115
133371
2007
65042
65186
93262
36966
17550
28010
77398
7270
130228
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