Association of Biochemical and Hematological Parameters With Enteric Fever Infection at the Dschang Regional Annex Hospital, Cameroon: A Cross-Sectional Study

Background Enteric fever is a systemic infection in humans caused by the Gram-negative bacilliSalmonella enterica serovars Typhi and Paratyphi. Although the diagnosis typically involves the isolation of Salmonella enterica serovars, it is often determined based on laboratory findings and clinical observations. However, due to the wide variety and the non-specific character of clinical features, making a definitive diagnosis presents numerous challenges. Therefore, the aim of this study was to find the predictive hematological and biochemical parameters which would serve in the diagnosis, prognosis, and treatment of typhoid fever cases. Methodology A cross-sectional study was conducted from November 2020 to September 2021 on1076consented volunteerparticipants. Stool culture and identification tests enabled us to distinguish three groups including 423 Salmonella Typhi positive patients, 115 S. Paratyphi positive patients, and 538 Salmonella negative participants. Biochemical and hematological parameters were evaluated using standard methods from commercial kits and Sysmex KX-21N automated hematology analyzer, respectively. A multiple logistic regression analysis was performed to identify the validity of the hematological and biochemical characteristics for enteric fever diagnosis. Results Multiple logistic regression showed hyper creatininemia, hypoalbuminemia, hyper total proteinemia, hyper alkaline phosphatase (ALP), hyper alanine aminotransferase (ALT), hyper total bilirubinemia, hyper conjugated bilirubinemia, hyper triglyceridemia, hyper C-reactive protein (CRP), leukopenia, thrombocytopenia, lymphopenia, monocytopenia, low hemoglobin, low hematocrit, low mean corpuscular volume (MCV), low mean corpuscular hemoglobin (MCH), low platelet, low platelet crit level, high platelet distribution width (PDW) level, high erythrocyte sedimentation rate 1 (ESR1) level as significant biological abnormalities associated (odds ratio {OR} > 1; p < 0.05) with enteric fever infection. Similarly, hyper ESR2 was an independent predictor (OR > 1; p < 0.05) of S. Typhi infection. However, a negative and significant association (OR < 1; p < 0.05) was recorded between enteric fever infection and high mean platelet volume (MPV). Conclusion Overall the results of the biochemical and hematological profiles can serve as potential diagnostic markers for typhoid fever. These markers can also be useful in the appropriate management of those with enteric fever, preventing severity and limiting outcomes of mortality.


Introduction
Enteric fever is a disease caused by ingestion of the Gram-negative bacilli Salmonella enterica serovar Typhi and Salmonella enterica serovar Paratyphi (S. Typhi and S. Paratyphi) through contaminated food and water. The most common symptoms of enteric fever are nausea, fever, abdominal pain, and vomiting. It is a common infectious disease in industrialized and developing countries [1]. About 11-20 million people are affected by typhoid fever each year, with 128,000-161,000 deaths globally, mostly in low-and middleincome countries whereas 6 million cases are caused by paratyphoid fever with 54,000 deaths each year worldwide [2]. The disease is endemic in many developing countries, especially in the Indian subcontinent, South and Central America, and Africa, with an annual crude incidence estimate of 130 per 100,000 personyears of observation [3]. Enteric fever remains one of the major public health and economic problems in developing countries with 2.6 times higher incidence of typhoid fever than the overall incidence in middleincome countries. Furthermore, due to poor access to drinking water and sanitation facilities, typhoid fever is a serious health problem in Cameroon [4].
The gold standard test, such as isolation of the bacterium by blood culture, stool culture, or uroculture is not readily available in hospitals and clinics in a resource-limited country like Cameroon. Additionally, selfmedication and abuse of antibiotics in the community often hinder the isolation of this bacterium through blood culture, stool culture, uroculture, and bone marrow culture, an invasive and difficult method. Hence, a serological test is widely used for the diagnosis of typhoid and paratyphoid fevers in most developing country laboratories. Unfortunately, the existing serodiagnostic assays such as Widal and Typhidot lack the required analytical sensitivity and specificity to diagnose typhoid fevers in disease-ravaging zones. Furthermore, requested serological assays are often misinterpreted due to the lack of appropriate control agglutination assays and deficient knowledge of assays' basic principles.
S. Typhi and Paratyphi cause localized infection of the gastrointestinal tract but can also multiply in the reticulo-endothelial cells and macrophages resulting in systemic infection. Hence, they affect one or more major organs of the body culminating in a wide range of organ infections such as hepatitis, gastroenteritis, myocarditis, endocarditis, and glomerulonephritis with or without associated acute renal failure or tubular necrosis [5]. This extended and severe infection may be multifactorial, involving endotoxin, local inflammatory, and/or immune reactions of the host. The development of encephalopathy, hemophagocytosis, anemia, leucopenia, gastrointestinal perforations, and acute respiratory distress syndrome (ARDS) are rare but represent serious complications of typhoid fever [6]. Hence, the incorporation of biochemical and hematological parameters is a source of potent biomarkers that would identify enteric fever and may also be utilized in the follow-up and management of victims, thus halting progression to bad prognosis and eventually mortality. The present study attempts to provide an in-depth analysis of different hematological and biochemical tests and associate each parameter with an enteric fever infection.

Design and study population
This study was a cross-sectional study conducted at Dschang Regional Annex Hospital, Cameroon, from November 2020 to September 2021. The study protocol was approved by the National Committee for Ethics in Human Health (CNERSH), Yaounde, Cameroon (ref: 2020/11/73/CE/CNERSH/SP). The participants were healthy individuals and those suffering from enteric fever. Male and female participants with no age restriction were selected for the study. Those included in the study were participants with or no symptoms of the disease, having signed the informed consent form and assent for those under 21. For non-inclusion criteria, the targets were those with a history of liver, kidney, and heart diseases; blood disorders; immunecompromised status; active alcohol consumption; pregnant women; positive serology for viral hepatitis; malaria; recent intake of a potential hepatotoxic drug; or any medication that may affect biochemical and hematological parameters. A total of 1082 participants were eligible for the study. Among them, subjects with hemolyzed blood serum and who did not provide complete information (n = 6) were excluded from the analysis. Finally, 1076 participants were retained for the study. Blood and stools were collected from participants who agreed to give their informed consent to participate in the study. Stool culture was used to stratify participants into sick and healthy and subdivided into 3 groups: 423 Salmonella Typhi positive patients, 115 Salmonella Paratyphi positive patients, and 538 Salmonella negative participants.

Stool and blood collection
Stools (25 g) were collected in wide-mouth plastic boxes from each participant and separated into two groups viz: Salmonella positive and negative patients. Blood (10 mL) was collected by venipuncture aseptically from the fasting participants at the elbow crease using an alcohol swab and a syringe fixed to a needle. Collected blood was separated into dry tubes to obtain serum and a tube with anticoagulant (EDTA) for evaluation of the hematological profile. The blood introduced in the dry tube was left to rest for 30 min at room temperature, then centrifuged at 3000 rpm for five minutes to obtain serum. The latter was separated from the clot into tightly screwed microfuge tubes and stored at -20 °C. These frozen sera were later analyzed for biochemical parameters.

Bacterial isolation and identification
Specimens were grown on Salmonella Shigella agar (SSA) and Hecktoen agar after pre-enrichment and enrichment. Then, the cultural and morphological characteristics of the isolates were studied and the isolates were duly identified following standard methods [7]. The serotyping of Salmonella enterica isolates was performed according to Kauffman-White Scheme [7]. Furthermore, polyvalent Salmonella antisera phase 1 and phase 2 flagellar H antigens were used for serovars determination of the Salmonella isolates.

Statistical analysis
The biochemical and hematological data were analyzed using statistical software for the social sciences (SPSS) version 22.0 (IBM Corp., Armonk, NY). The results were expressed as mean ± standard deviation. The means of each biochemical parameter in different groups were compared using the Waller-Duncan test when significant differences were detected by ANOVA. Waller Duncan's conformity test was used to compare the means of the group variables to the reference values. Cut-off values provided by the kits were used to classify the parameter as abnormal (value out of the reference range) and normal (value within the reference range). Categorical variables were described as numbers and percentages. A Chi-square test was performed to compare the frequencies of biochemical abnormalities in the three groups. A multivariate logistic regression analysis was performed to determine true supportive parameters in enteric fever diagnosis. A p < 0.05 was considered significant.

Results
Creatinine, urea, uric acid, alkaline phosphatase (ALP), total bilirubin, conjugated bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides, total protein, and C-reactive protein levels were found to be higher in patients with both Salmonella Typhi and Paratyphi compared to the control group ( Table 1). On the contrary, the mean values of albuminemia, total cholesterol, LDLcholesterol, and HDL-cholesterol were significantly decreased (p < 0.05) in Salmonella Typhi and Paratyphipositive patients compared to the control group. The increased levels of uremia, uricemia, ALT, total proteins, and C-reactive protein (CRP) in positive patients with typhoid fever were higher than in those with paratyphoid fever. The mean values of all biochemical parameters in healthy participants were within the normal ranges. However, the mean values of creatininemia, uremia, albuminemia, ALP, total bilirubin, conjugated bilirubin, AST, triglycerides, total protein, C-reactive protein (in both S. Typhi and Paratyphi positive patients) and ALT (in solely S. Typhi positive patients) were out of the normal ranges.  In S. Typhi and S. Paratyphi positive patients, the mean values of erythrocytes, mean corpuscular hemoglobin concentration (MCHC), erythrocyte sedimentation rates 1 and 2 (ESR 1 and 2) were significantly (p < 0.05) increased compared to the negative group (    Creatinine, a byproduct of creatine phosphate in the muscle is entirely eliminated by the kidney. A simultaneous increase in creatinine and urea in most patients with enteric fever indicates renal dysfunction. This can be linked to an increased catabolic rate which characterizes the disease. In addition, renal impairment can be explained by immune complex deposits and toxin-induced nephropathy, which is a direct result of the toxin effect on the podocytes [9]. The raised urea and creatinine levels in enteric fever have been reported [10]. On the contrary, urea was observed to be non-affected in patients with enteric fever in another study [11]. The liver is commonly implicated in patients with typhoid and paratyphoid fevers. In this study, we observed hyper alkaline phosphatase (62.4%), hyper ALT (79.2%), and hyper total bilirubin (66.0%), which are quite higher than those already reported: 44%, 73.3%, 30.6%, respectively [12]. A previous study conducted in India showed a significant increase in the levels of ALT, AST, PAL, total and conjugated bilirubin as well as a significant decrease in albumin levels [13]. Increased hepatic enzymes is suggestive of liver damage. This can be due to specific endotoxin effects locally or systemically, inflammatory reactions to ulceration of intestines, and S. Typhi cytotoxin production in infected Kupffer cells or liver macrophages [14]. High total bilirubin (jaundice) in enteric fever patients is a result of hemolysis and in severe conditions can lead to liver injury. Additionally, hyper-bilirubinemia observed in patients with enteric fever could be due to the multiplication of S. Typhi/Paratyphi within the biliary tract which causes clogging of the biliary flow [15]. The incidence of jaundice and raised bilirubin levels in enteric fever have been reported [14]. In the current investigation, fever and jaundice were the presenting symptoms in 66.0% which is quite higher than that obtained by earlier investigators (30.6%) [12]. Our results demonstrate that liver involvement in enteric fever is usually associated with extrahepatic complications. Generally, hypoproteinemia is known to be associated with complications and mortality in patients with acute infectious disease [16]. Albumin and total proteins were also significantly lower in enteric fever patients compared to normal individuals. This result disagrees with the findings of earlier investigators who showed an increase in albumin in chronic typhoid patients [17]. Nevertheless, suggestive mechanisms for the drop of plasma proteins have been proposed thus: large vascular leakage of plasma proteins owing to raised capillary permeability, reduced protein intestinal absorption due to low ingestion, albumin clearance in urine, elevated protein catabolism, and diminished liver synthesis due to hepatic damage [18]. Additionally, proteins can be lost as a result of kidney dysfunctions which lead to an excessive amount of proteins being excreted in the urine.
The lipid profile is known to alter in patients with severe sepsis; hence, the evaluation of the lipid profile could be necessary for performing a typhoid fever diagnosis. In this study, patients with enteric fever showed a significant increase in the level of triglycerides compared to their controls. This finding agreed with that of a previous study which showed an elevation in the levels of triglycerides in patients with enteric fever [19]. This was unlike another study that showed hypocholesterolemia and hyper LDL-cholesterolemia as well as a non-significant change in triglyceride and HDL-cholesterol levels in typhoid fever [20]. This disparity in results may be due to differences in analytic methods used and the extent of illness in a patient as well as the patient's dietary intake. The increase in triglyceride levels can be explained by increased biosynthesis during infection [21]. High levels of triglycerides in the bloodstream have been linked to atherosclerosis which increases the risk of heart disease and stroke.
Although the C-reactive protein (CRP) test is non-specific, it is very useful in the characterization of acute inflammation and infection [22]. The inflammatory conditions and host responses to infection cause the release of interleukin-6 and other cytokines that trigger the synthesis of CRP and fibrinogen by the liver [22]. Hence, we suggest using CRP in enteric fever detection given the significant and positive relationship (OR > 1; p < 0.05) between high levels of CRP and enteric fever.
The results of our investigation also revealed positive and significant (OR > 1; p < 0.05) relationships between enteric fever infection and leukopenia, thrombocytopenia, lymphopenia, monocytopenia, low hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and plateletcrit levels as well as high platelet distribution width (PDW) and erythrocyte sedimentation rate 1 (ESR1) levels. Similarly, higher ESR2 was an independent predictor of S. Typhi infection. Nonetheless, a negative and significant association (OR < 1; p < 0.05) was recorded between enteric fever infection and high mean platelet volume (MPV). This observation is indicative that hematological abnormalities can reinforce a positive enteric fever condition and also contribute to disease management. Enteric fever is a multisystem disease that activates bone marrow which results in an increase in MPV, PDW, and ESR. ESR measures nonspecific inflammation. The significant increase in ESR in infected patients may be due to an increased inflammatory response. The decrease in the levels of some hematological parameters (WBC, platelet, lymphocytes, monocytes, hemoglobin, hematocrit, MCV, MCH, and plateletcrit) may be a result of declined bone marrow activity and hemophagocytosis. The latter is the main route of elimination of the pathogen in victims of enteric fever [23]. Our results are consistent with those of a previous report which showed that most patients with typhoid fever are moderately anemic, and have leukopenia, thrombocytopenia, and relative lymphocytopenia [23]. Thrombocytopenia was reported as the most common complication of enteric fever [23] whereas leukopenia is considered a common hematological finding in enteric fever [12].
In this study, leukopenia was observed at least in 82.5% of cases, which is quite higher than the 11.2% previously reported [24]. Leucocytosis in children is common within 10 days of sickness and in those experiencing bleeding [25]. However, in the current study, leucocytosis was seen in less than 3% of patients with enteric fever. The low hematocrit and hemoglobin levels recorded in patients with typhoid/paratyphoid fever indicate that anemia could be involved in at least 63.0% of patients. A previous study documented anemia in 48.0% of patients with enteric fever [26]. Hemolysis, elements of gastrointestinal blood loss, and transient marrow suppression have been cited as the mechanisms of anemia during enteric fever [24]. Low MCV means that red blood cells (RBC) are smaller than normal and may indicate microcytic anemia. This condition may be caused by iron deficiency. The mean corpuscular hemoglobin concentration (MCHC) levels were abnormally low in the majority of the patients, suggesting microcytic hypochromic anemia. This result is discordant with those of an earlier study which showed that enteric fever was associated with normocytic normochromic anemia [27]. The hematological and biochemical changes observed in this study can be elucidated by multiple organ implications in enteric fever with hallmarks of bacteremia and sustained normal fever. Few studies have been performed on hematological and biochemical profiles of enteric fever patients in Cameroon, but no study report is available yet on hematological profiles in our region.

Conclusions
Overall, our results reveal high elevation and diminution of certain biological factors which could be exploited for healthcare benefits in conditions of enteric fever. Our study shows that enteric fever is associated with hyper creatininemia, hypoalbuminemia, hyper total proteinemia, hyper ALP, hyper total bilirubinemia, hyper conjugated bilirubin, hyper ALT, hyper triglyceridemia, hyper CRP, leukopenia, thrombocytopenia, lymphopenia, monocytopenia, low plateletcrit, high platelet distribution width (PDW) level, and high erythrocyte sedimentation rate. Moreover, this study brings to light a high prevalence of microcytic hypochromic anemia in the majority of patients with enteric fever. Our findings emphasize the importance of using these biochemical and hematological markers to enhance serological diagnosis and for the appropriate management of enteric fever patients.

Additional Information Disclosures
Human subjects: Consent was obtained or waived by all participants in this study. Cameroon National Committee for Ethics in Human Health (CNERSH), Yaounde, Cameroon issued approval 2020/11/73/CE/CNERSH/SP. The Cameroon National Committee for Ethics in Human Health (CNERSH) in its session of November 11, 2020, examined the research project entitled "Evaluation of biochemical and hematological abnormalities in patients with Salmonella enterica and study of genetic mutations associated with antibiotic resistance" submitted by Tito Aloys Ndima Etouke, Ph.D. candidate at the Faculty of Science of the University of Dschang. This Research project is of certain scientific interest and does not present any risk to the participant. The objectives and methodology of the study are clearly described. The principle of data confidentiality is respected. The skills required for the supervision of research work are presented. In view of the above, CNERSH approves the implementation of this version of the protocol for a period of one year. Tito Aloys Ndima Etouke is responsible for the scrupulous respect of the protocol and should not make any amendment, however minor, without the favorable opinion of CNERSH for the monitoring of the ethical aspects of the approved protocol. The final report of the research project must be submitted to CNERSH/SP for archiving. This ethical clearance is issued to serve and assert its rights. It can be cancelled in the event of non-compliance with the regulations in force and the aforementioned recommendations. Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue.

Conflicts of interest:
In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.