RESEARCH PROJECT PROPOSAL
ON
SERUM ELECTROLYTES/ UREA/ CREATININE IN PATIENTS WITH SALMONELLA TYPHI AND PARATYPHI INFECTIONS
BY
NIGERIAN UNIVERSITIES RESEARCH TOPICS
(CHEMICAL PATHOLOGY SPECIATY)
JANUARY, 2017.
INTRODUCTION
1.1 BACKGROUD OF STUDY
The genus salmonellae consists of two species: (1) S. enterica which is divided into six subspecies: S. enterica subsp. enterica, S. enterica subsp. salamae, S. enterica subsp. arizonae, S. enterica subsp. diarizonae, S. enterica subsp. houtenae and S. enterica subsp. indica; and (2) S. bongori (formerly called S. enterica subsp. bongori). The subspecies name does not need to be indicated, as only serovars of subspecies enterica has a name. Therefore we write e.g. Salmonella Typhimurium.
Salmonella is generally identified as being a non-lactose fermenting, (NLFs) Gram negative rod shaped organism, ranging 0.7 to 1.5 x 2 to 5 μm in size. With the exception of S. Pullorum and S. Gallinarum, they are motile with peritrichous flagellae. D-glucose is fermented with the production of acid and usually gas. Other carbohydrates usually fermented are L-arabinose, maltose, D-mannitol, D-mannose, L-rhamnose, D-sorbitol (except ssp VI), trehalose, D-xylose and dulcitol.
Salmonella is oxidase negative, catalase positive, indole and Voges Proskauer (VP) negative, methyl red and Simmons citrate positive, H2S producing and urea negative. Some of these characteristics are used for biochemical confirmation of Salmonella (ISO 6579, 2002).
Typhoid and paratyphoid fevers are commonly grouped together under the collective term 'enteric fever'. Typhoid is caused by Salmonella typhi (strictly termed S. enterica sub-species enterica serotype typhi) ( Sethuraman et al. 1994,) and paratyphoid is caused by either Salmonella paratyphi A, B, or C. Serologically S. typhi is LPS antigen 09, 12, protein flagellar antigen Hd, and capsular polysaccharide antigen Vi positive. Salmonella paratyphi B is also known as S. schottmuelleri, and Salmonella paratyphi C as S. hirschfeldii.
These Salmonelloses are highly adapted infections of man, having no animal or environmental reservoir. Occasionally other Salmonellae may cause an enteric fever-like syndrome. The complete genome of S. typhi CT 18 has been sequenced recently (Parkhill et al, 2001).
All isolates of S. typhi are closely related, although the genome is remarkably plastic (with homologous recombination around 7 ribosomal RNA genes) (McClelland, et al). Typhoid fever was once a major cause of morbidity and mortality throughout the world with a case fatality rate of approximately 15% (Christie AB. 1984; Hoffman et al, 1986).
Over the past century the infection has been largely eradicated from more affluent temperate countries, although it remains a major infection in tropical areas of the world (Haffman et al, 1984). Precise estimates of the mortality and morbidity of infectious diseases in poor countries are notoriously unreliable, but estimates suggest that there are approximately 16,000,000 cases of typhoid each year, with 600,000 deaths (Ivanoff B; 1995).
1.2 Epidemiology
Typhoid and paratyphoid are transmitted mainly by the fecal-oral route. In most cases an asymptomatic carrier of S. typhi, or an individual who has recently recovered from the infection, continues to excrete large numbers of organisms in the stool and contaminates food or water, either through direct food handling, through transfer of bacteria by flies and other insects, or by contamination of potable water (lin. et al 200; Luby et al 1998; Luxemburger et al, 2001).
Approximately 10% of patients recovering from typhoid fever excrete S. typhi in the stool for three months, and in the past 2-3% became permanent carriers. These infections have great potential for epidemic spread therefore (Soe GB, Overturf GD, 1987; Thong et al, 1994). In the tropics enteric fever tends to be more common during the hot dry seasons when the concentration of bacteria in rivers and streams increases, or in the rainy season if flooding distributes sewage to drinking water sources.
In some areas the incidence of typhoid may be as high as 1,000 cases per 100,000 population per year. In such areas typhoid is predominantly a disease of children, and stool excretion of S. typhi during and after infection is the main source of the infection. In such areas Salmonella typhi infections are commonly mild and self limiting. Severe disease represents the "tip of the iceberg". In temperate countries persistent carriers are a more important reservoir of infection (Haffman SI; GT Strickland, 1984). For travellers the highest attack rates are associated with visits to Peru [17 per 105 visits], India [11/105 visits], and Pakistan [10/105 visits].
Although Indonesia has a reported annual incidence up to 1%, the attack rate for travellers is low. In general the mortality of enteric fever is low (< 1%) where antibiotics are available, but in poorer areas, or in the context of natural disasters, war, migrations, large concentrated refugee populations, and other privations, the mortality may rise to 10-30%, despite antibiotic therapy.
Typhoid tends to cluster in families (Luxemburger et al, 2001), presumably reflecting a common source of the infection and is associated with poverty and poor housing. Apart from exposure to the contaminated food (often ice creams or iced drinks) or water source, a number of host factors increase the risk of Salmonella infections. Disease related (achlorhydria) or iatrogenic (antacids, H2 blockers, proton pump inhibitors) reduction in stomach acidity or gut pathology (surgery, inflammatory bowel disease, malignancy) and recent antibiotics increase the susceptibility to infection.
Disease related or iatrogenic immunosuppression and several other infections, notably schistosomiasis, malaria (Luby et al 1998; Luxemburger et al, 2001), histoplasmosis and bartonellosis, are associated with an increased risk of Salmonella infections. Typhoid is more common, and more severe at the extremes of age. Neonatal typhoid usually acquired from the mother may follow a fulminant course often with meningitis (Bhutta ZA, 2000;Blutler et al, 1991). Patients with hemoglobinopathies, particularly sickle cell disease, are also at increased risk.
1.3 Clinical Manifestations
The clinical features of enteric fever vary considerably between different geographic regions. In many areas typhoid becomes the leading differential diagnosis of a patient with a fever which has lasted for more than one week. The clinical features of typhoid and paratyphoid fever are generally similar, although paratyphoid tends to be a more mild infection (Christie, 1984).
Most patients with enteric fever present with a non-specific gradual onset of an influenza-like illness although Salmonella typhi infection can present with fever and a bewildering array of signs and symptoms ranging from non-metastatic central nervous system syndromes including psychosis and cerebellar ataxia (Trevett et al, 1994), through to focal involvement of bone (Declereq et al 1994), liver (El Newihi et al, 1996; Jagadish et al, 1994; Schwartz et al, 1994), spleen (Allal et al, 1993), testes (Jones et al, 1982), meninges (Lecour et al, 1994;Sharma et al, 1992), vascular prostheses, atheromatous plaques etc., (Van Basten JP, Stockenbrugger R, 1960).
In general the enteric fevers are sub-acute infections with an incubation period of approximately 7 - 14 days (range 3-60 days) following exposure. The illness begins insidiously with non-specific signs and symptoms of fever (Richens et al, 1992), headache, muscle and joint aches, malaise, lassitude, anorexia, often a dry cough (sometimes associated with a sore throat) (Sharma et al, 1992).
The spleen enlarges, but lymphadenopathy is not usually prominent. There may be a few rose spots (sparse, pink, macular papular lesions which blanche with pressure and fade after two or three days) on the thorax or abdomen (usually less than 10), but these are often unnoticed (particularly in dark skinned patients). In paratyphoid fever rose spots may be more prominent.
The classic "stepladder fever" of typhoid is unusual although the fever does become higher as the disease progresses, until it levels fluctuating between 39°C and 40°C. Mild chills and sweating are common but true rigors are rare. Relative bradycardia is considered common in typhoid although in many series this has not been a feature of the disease.
Some abdominal complaints are usual although either diarrhoea or constipation may occur. There is usually some abdominal discomfort, and even in the first week of the disease the patient may notice passage per-rectum of a small amount of blood or melena. Normal bowel habit is unusual in typhoid. Diarrhea (Roy et al, 1985) is more common in infants (Bhutta et al, 1991), and in patients with AIDS. Constipation occurs in approximately 40% of patients. A fulminant onset with a septic shock presentation may occur but is unusual.
The clinical evolution of untreated typhoid is divided classically into weeks (Christie AB, 1984). During the first week the fever rises gradually, and in the second week reaches a high plateau. By the second week the patient has become progressively weaker, has lost weight, and has often developed the characteristic effect from which typhoid derives its name (typhoid means "like typhus", which in turn derives from the Greek typhos meaning smoke, and refers to the clouding of the sensorium in these infections).
The patient remains apathetic or depressed, anergic, often confused and withdrawn whilst lying in bed, yet sleep does not come easily. By the third week of infection, if untreated, a dangerous stage is entered upon in which either intestinal perforation or hemorrhage become more likely as the necrotic Peyer's patches either erode through the wall of the terminal ileum (Bitar R, Tarpley J, 1985;Butler et al, 1991- 1985), or penetrate a large blood vessel. In large series reported before the pre-chloramphenicol era intestinal hemorrhage occurred in 7-21% of cases and intestinal perforation in between 0.7 and 4.7% of cases (Christie AB, 1984).
In the antibiotic era the incidence of perforation has fallen slightly to approximately 3% of cases, and clinically significant intestinal bleeding now occurs in less than 2% although figures still vary considerably from series to series. The risk of both hemorrhage and perforation increase from the middle of the second week.
In the third week of the illness the patients is often withdrawn, obtunded, or intermittently delirious. The abdomen becomes distended and there may be, vomiting, and abdominal pain. Right upper quadrant pain may indicate cholecystitis or cholangitis (3% of cases) whereas lower quadrant pain with signs of peritoneal irritation may indicate perforation.
Complications in the third and fourth week also include pneumonia (Parry et al, 2002) ARDS (Buczka GB, McLcan JN, 1994), the development of acute psychosis, coma (147,26), myocarditis (Prabha et al, 1995), pericarditis, orchitis (Zafar et al, 1995), venous thrombosis (Ghosh JB, Samanta S, 1994), splenic rupture (Ali et al, 1994), meningitis (Lecour et al, 1994), hepatic dysfunction (Jagadish et al, 1994) and occasional renal failure (Sitprija et al, 1974). If the patient survives this phase of the illness there follows a gradual recovery.
As the duration of infection is an important determinant of the risk of severe complications, a delay in receiving appropriate antibiotic treatment may have serious consequences. In some endemic areas multi-drug resistance (and thus delayed treatment with effective antimicrobials) has led to an increase in mortality, particularly in infants. (Bhutta ZA, 1996).
1.4 Treatments
Control requires treatment of antibiotics and vaccines prescribed by a doctor. Major control treatments for paratyphoid fever include ciprofloxacin for ten days, ceftriaxone/cefotaxime for 14 days, or azithromycin.
1.4.1 Prognosis
Those diagnosed with Type A of the bacterial strain rarely die from it save in rare cases of severe intestinal complications. With proper testing and diagnosis, the mortality rate falls to less than 1%. Antibiotics such as azithromycin are particularly effective in treating the bacteria (WHO, 2008)
1.4 JUSTIFICATION
The present study is to estimate the serum urea, creatinine and electrolyte status of patients presenting with acute gastroenteritis. Sixty patients who presented to Kathmandu Medical College and Teaching hospital from 15 June to 15 July 2005 with acute diarrhea with or without associated vomiting, causing dehydration severe enough to require hospital admission were investigated for serum urea, creatinine and electrolyte level.
Out of 60 patients investigated, serum sodium and potassium level were available for 34 patients. Only one (2.9%) patients had sodium level below 135mEq/l, thirty two (94.11%) had sodium level between 135-146 mEq/l and one (2.9%) had sodium level above 146mEq/l. Similarly 9 (26.47%) patients had potassium level below 3.5mEq/l, 22 (64.70%) patients had potassium level between 3.5-5 mEq/l and 3 (8.82%) patients had level above 5 mEq/l.
Serum urea and creatinine level were available for 47 patients. 36 (76.59%) patients had serum urea level between 15-45mg/dl and 11 (23.40%) patients had urea level above 45 mg/dl. 35 (74.46%) patients had serum creatinine level between 0.5-1.4 mg/dl and 12 (25.53%) had serum creatinine level above 1.4 mg/dl. In this study hypokalaemia was noticed more than hyponatremia and significant number of patients also showed increased level of serum urea and creatinine. Therefore, serum urea, creatinine and electrolytes should be closely monitored in patients with acute gastroenteritis (KcM, et al, 2006).
1.4 RESEARCH QUESTIONS
This study will provide answers to the following questions:
Is there any significant change in serum E/U/Cr or any of this parameter in patients with salmonella typhi and paratyphi?
Does the level of salmonella typhi and paratyphi contribute to the increase and decrease of E/U/Cr?
Can salmonella typhi and paratyphi be rule out or involve in diagnosis of renal function?
1.5 RESEARCH HYPOTHESIS
Null hypothesis (HO):
There could not be a significant change in serum E/U/Cr of patients with salmonella typhi and paratyphi when compare with control samples.
The level of salmonella does not have significant increase in serum E/U/Cr or any of this parameter when compare with the control samples.
Salmonella typhi and paratyphi infections should be rule out in renal function diagnosis.
Alternate hypothesis (H1):
There could be a significant change in serum E/U/Cr of patients with salmonella typhi and paratyphi when compare with control samples.
The level of salmonella typhi and paratyphi could have significant increase in serum E/U/Cr or any of this parameter when compare with the control samples.
Salmonella typhi and paratyphi should be put into consideration in diagnosis of renal function
1.6 AIM OF STUDY
The aim of this study is to evaluate serum electrolytes, urea, and creatinine in patients infected with and without salmonella typhi and paratyphi, (Etsako West Local Government Area, Auchi, Edo State as a case study).
1.7 SPECIFIC OBJECTIVES
1. To evaluate serum electrolytes (Na+, Cl-, K+, and HCO3-) of salmonella infected patients and compare with control samples, (Etsako West Local Government Area, Auchi, Edo State as a case study).
2. To evaluate serum urea of salmonella infected patients and compare with control samples (Etsako West Local Government Area, Auchi, Edo State as a case study).
3. To evaluate serum creatinine of salmonella infected patients and compare with control samples, (Etsako West Local Government Area, Auchi, Edo State as a case study).
2.0 MATERIALS AND METHODS
2.1 AREA OF STUDY
This study will be carried out in Etsako West Local Government area, Auchi, Edo state. It is a Local Government Area of Edo State, Nigeria. Its headquarter is in the town of Auchi. Etsako West is made up of four clans; Uzairue, Auchi, South Ibie, Awain clan and Agbede. The major town in this LGA includes Auchi, Jattu, Agbede, Aviele, Iyamoh, Jagbe, and Awain clan (Ewora, idegun, Ama, Ibhioba). It has an area of 946km2 and a population of 197609 at 2006 census. It also has the prestigious Federal Polytechnic attended by students all over the state and recently Edo State University Iyamoh. The residents are mainly farmers and their source of water is rivers, wells and few boreholes. The postal code of the area is 312. (‘post offices- with map of LGA ‘. NIPOST. Archive from the original on October 7, 2009 -10-20).
2.2 POPULATION OF STUDY
The study will be conducted on 100 individuals; comprising of 50 individual infected with salmonella typhi and paratyphi and 50 individual without infection (controls) attending public and private health facilities and as well as their residential home in Etsako West Local Government Area, Auchi, Edo State.
2.3 RESEARCH DESIGN
The research is designed to assess E/U/Cr in patients infected with salmonella typhi and paratyphi in Etsako West Local Government Area, Auchi, Edo State. This study shall be carried out within three (3) months.
2.4 INCLUSION CRITERIA
Only individuals that are infected with salmonella typhi and paratyphi will be recruited for this study.
2.5 EXCLUSION CRITRERIA
Salmonella individual with other complications such as malaria, hypertension, diabetes, tuberculosis, HIV/AIDS etc. will be excluded from this study.
2.6 SAMPLE COLLECTION AND SEPARATION
Two plain bottles will be use for each subject. Whole blood sample (5ml) will be collected by clean venopuncture from the ante-cubital fossa into already labeled plain bottle, without undue pressure to either the arm or the plunger of the syringe. The blood sample collected into the plain bottle will be allow to clot and then centrifuge at 3000 rpm for 5 minutes to obtain serum. The serum supernatants will be separated into sterile plain bottle and label. A portion of the obtained serum will be use to test for the present of salmonella typhi and paratyphi, while the remaining serum will be stored frozen until analysis is done at room temperature.
2.7 SAMPLE ANALYSIS.
2.7.1 DETERMINATION OF SALMONELLA TYPHI AND PARATYPHI TITRE IN THE SERUM SAMPLE USING WIDAL METHOD.
principle of widal test
Salmonella typhi and Salmonella paratyphi A, B and C cause enteric fever (typhoid and paratyphoid) in human. Widal test is a common agglutination test employed in the serological diagnosis of enteric fever. This test was developed by Georges Ferdinand Widal in 1896 and helps to detect presence of salmonella antibodies in a patient’s serum.
Patients infected with Salmonella produce antibodies against the antigens of the organism. Antibodies in serum, produced in response to exposure to Salmonella organisms will agglutinate bacterial suspension which carries homologous antigens. This forms the basis of Widal test.
The organisms causing enteric fever possesses two major antigens namely somatic antigen (O) and a flagellar antigen (H) along with another surface antigen, Vi. During infection with typhoid or paratyphoid bacilli, antibodies against flagellar antigen of S. typhi (H), S. paratyphi A (AH), S. paratyphi B (BH) and Somatic Antigen of S.typhi (O) usually become detectable in blood, 6 days after the onset of infection.
Those antigens specifically prepared from organism are mixed with patient’s serum to detect the presence of antibodies. Positive result is indicated by the presence of agglutination. Absence of agglutination indicates a negative result. The paratyphoid O antigens are not employed as they cross react with the typhoid O antigen. If agglutination occurs with O antigen then it is considered positive for Salmonella typhi. If agglutination occurs in A or B antigen then it is confirmed as positive for Salmonella paratyphi. Agglutination will occur in H antigen for all the cases of antigens like O, A, and B.
PROCEDURE OF WIDAL TEST
The Widal test will be carry out using the slide agglutination method, it is divided into two steps:
1. Qualitative Slide Test.
2. Quantitative Slide Test
Qualitative Slide Test
Procedure:
1. Bring all reagents to room temperature and mix well.
2. Add 1 drop of test sample (25µl) into each reaction circle labeled as O, H, AH, BH according to given antigen solution.
3. Add 1 drop of positive control (25µl) into the circle marked as PC and 1 drop of negative control (25µl) into the reaction circle marked as NC.
4. Add antigen solutions of Salmonella typhi ‘O’, Salmonella typhi ‘H’, Salmonella paratyphi ‘AH’ and Salmonella paratyphi ‘BH’ to circles labeled as O, H, AH, BH respectively in which test samples has been added.
5. Mix it thoroughly with the aid of applicator stick and rotate the slide gently.
6. Observe for agglutination.
Interpretation:
· Positive Test : Agglutination within a minute
· Negative Test : No agglutination
Quantitative Slide Test
This is performed for the samples which showed positive agglutination during qualitative test.
Procedure:
1. Bring all reagents to room temperature and mix well.
2. Dispense one drop of saline into the first reaction circle and then place 5, 10, 20, 40, 80 ul of the test sample on the remaining circles.
3. Add a drop of the antigen, which showed agglutination with the test sample in the screening (qualitative) method, to each circle.
4. Mix the contents of each circle with the aid of applicator stick and rotate the slide gently.
5. Observe for agglutination.
Interpretation:
The antibody titre of the test sample is its highest dilution that gives a visible agglutination. 80 µl corresponds to 1 in 20 dilution, 40 µl to 1 in 40, 20 µl to 1 in 80, 10 µl to 1 in 160 and 5 µl corresponds to 1 in 320 titre. Agglutinin titre greater than 1:80 is considered as significant infection and low titres indicate absence of infection.
LIMITATIONS OF WIDAL TEST
1. Tests done within 7 days of illness and after 4 weeks are usually negative.
2. The local titre of the place should be known for the results interpreted correctly.
3. This test (Quantitative) is highly time consumable.
4. Previous typhoid vaccination may contribute to elevated agglutinins in the non-infected population.
5. Other infections of non-enteric salmonella infection such as Typhus, Immunological disorders, chronic liver disease may cause false positive reaction.
6. Cross reaction between malaria parasites and salmonella antigens may cause false positive Widal agglutination test (Dhurba Giri, 2016).
2.7.2 CREATININE ANALYSIS
Serum creatinine will be analysed spectrophotometrically or colorimetrically using the Jaffe-Slot modified alkaline picrate colorimetric method.
Principle of the Jaffe-Slot alkaline picrate
Creatinine method
Creatinine reacts with picric acid in an alkaline medium. The absorbance of the yellow-red colour produced is measured in a colorimeter using a blue green filter 490nm (Ilford No. 603) or in a spectrophotometer at 490 nm wavelength.
2.7.3 UREA DETERMINATION
Serum urea will be determined using the urease-berthelot reaction.
Principle of the urease-Berthelot reaction
This uses the enzyme urease to hydrolyze urea at 37 °C. The ammonia produced reacts with alkaline hypochlorite and phenol in the presence of a catalyst to form indophenols which is measured at 570 nm. (Cheesbrough,1998).
2.7.4 ELECTROLYTES ANALYSIS
Ion selective electrode will be use for the determination of serum sodium, potassium, chloride and bicarbonate.
Principle of operation
The sample is mixed with the high ionic strength ISE electrolyte buffer and passed through the flow cell, a potential is generated at the surface of the ion selective electrodes and thereby measure the sodium, potassium, chloride concentration present in the sample (Tietz, 2014).
2.8 DATA ANALYSIS
Data obtain will be analyze using SPSS version 17 statistical soft ware package. Results generated will be expressed as mean ± SD and a p-value of ˂0.5 will be considered significant.
FINANCIAL PLAN
S/N
|
ITERMS
|
QUANTITY
|
AMOUNT (₦)
|
1.
|
Plain containers (sample containers).
|
3 Packs
|
4,500
|
2.
|
5mls syringes and 2mls extra needles.
|
2 packs each
|
5,500
|
3.
|
Cotton wool, hand gloves and methylated spirited
|
2,500
|
|
4.
|
Widal kit (CJ. SMART)
|
2 packs
|
20,000
|
5.
|
Samples analysis (E/U/Cr)
|
1 each
|
2,500
total of 100 = 250,000
|
6.
|
transportation
|
7,000
|
|
7.
|
Sourcing information from internet and library
|
10,000
|
|
8.
|
Typing, printing, photocopying and binding
|
25,000
|
|
9.
|
Miscellaneous
|
15,000
|
|
TOTAL
|
339,500
|
WORK PLAN
S/N
|
ACTIVITIES
|
DURATION
|
1.
|
Collection of information/ ethical Approval/ lab access
|
2 weeks
|
2.
|
Collection of samples
|
3 weeks
|
3.
|
Analysis of samples
|
2 weeks
|
4.
|
Analysis of results and compilation of works
|
3 weeks
|
5.
|
Typing, printing and binding of work
|
2 weeks
|
TOTAL
|
12 weeks
|
REFERENCES
ISO 6579 :2002(E) 4rd ed. Microbiology - General guidance on methods for the detection of Salmonella, International Organization for Standardization, Geneve, Switzerland.
(http://laboratoryinfo.com/widal-test-principle-procedure-result-interpretation-and-limitations/)Posted on January 28, 2016 by Dhurba Giri in Bacteriology, Microbiology // 13 Comments
"Water-related Diseases." Communicable Diseases 2001. World Health Organization. 31 Oct 2008 <http://www.who.int/water_sanitation_health/diseases/typhoid/en/
Kc M, Gurubacharya DL, Lohani R, Rauniyar A. Serum urea, creatinine and electrolyte status in patients presenting with acute gastroenteritis. Kathmandu Medical College and Teaching hospital 2006 Jul-Sep;45(163):291-4.
Cheesbrough, M. (1999): District laboratory practice in tropical countries. Part 1. Low price edition. Cambridge University press.UK. pp 333&337
Carl .A. Burtis, David E. Bruns Tietz (2014) Foundament of clinical chemistry and molecular Diagnosis,seventh edition. Indian. 24: 415.
Prabha A, Mohanan, Pereira P, Raghuveer CV. Myocarditis in enteric fever. Indian J Med Sci 1995;49:28-31. [PubMed]
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