Identi cation of Leptospira in Patients With Renal Failure Using Serological, Molecular and Pathological Based Techniques, in Shiraz, Iran

Leptospirosis is a relatively rare bacterial infection that affects people and animals caused by pathogenic species of Leptospira. The present study was conducted using nested polymerase chain reaction (PCR), microscopic agglutination test (MAT) and hematological and biochemical tests on 200 blood samples of renal disorders patients in Shiraz, Iran. Also nested-PCR assay and Warthin-Starry (WS) silver staining method was performed on 30 nephrectomised kidney sample. The frequency of pathogenic species of Leptospira infection in patients with renal disorders was 20 % and this infection was signicantly correlated with BUN, anemia, RDW, MCV, MCH and hemoglobin levels (P < 0.01). MAT analysis showed that serum samples had positive titers against L. Grippotyphosa (13 samples), L. Ballum (6 sample), L. Pomona (3 samples), L. Canicola (2 samples), L. Icterohaemorrhagiae (1 sample) and L. Hardjo (1 sample) serovars. Twenty-three percent of the kidney samples from the patients with pyelonephritis were infected with the pathogenic species of Leptospira. This study showed that pathogenic Leptospira serovars are present in this area and in patients with renal disorders more attention should be paid to this zoonotic disease.


Introduction
Leptospira is consider as one of the causative agents of acute febrile infection both in human and animals. Urine of the clinically infected and carrier animals are the major sources of human infection (Roger et al., 2006). Currently, the genus Leptospira is divided into 35 distinct species classi ed into three phylogenetic clusters, the classi cation is which hypothetically associate with the pathogenicity of the bacteria (Thibeaux et al., 2018a;Thibeaux et al., 2018b;Vincent, 2019). Leptospira species are considered ubiquitous, as they are usually present in a wide variety of environments such as surface water, soil, with the wide host range including mammals but also in birds, amphibians, and reptiles (Dietrich, et al., 2015;Jobbins et al., 2015). According to the phase of infection, clinical signs of leptospirosis are varied from the acute icteric fatal infection to the chronic anicteric symptoms (Levett et al, 2001). In human, the disease is a career associated infection repeatedly occurred in farmers, veterinarian and abattoir workers (Thaipadungpanit et al., 2007;Sharma, 2008) with the major risk factors such as water sports and travel to the endemic countries (Victoriano et al., 2009) using nested PCR assay and restriction length polymorphism (RFLP) techniques revealed a good correlation between rejected kidneys and infection with Leptospira, in Shiraz slaughterhouse (Taghadosi et al., 2016). In Iran, the disease in human was rstly reported by Rafyi and Maghami in 1968. While, in the developed countries, Leptospira infection was not frequently associated with the acute renal failure (ARF) (Jayakumar et al., 2006;Regina et al., 2008).
The microscopic agglutination test (MAT) is frequently applied as a standard screening test all around the world (Regina et al., 2008;Abdollahpour et al., 2009). The standard MAT was established according to the reaction of the bacterial cell components and hyper immune antisera. A PCR assay using species-speci c primers is also developed for the quick identi cation of leptospiral DNA (Rahelinirina et al., 2010;Thaipadungpanit et al., 2011;Vital-Brazil et al., 2012;Benacer et al., 2013;De Brito et al., 2018). Various diagnostic techniques such as direct culture, selective staining and immunohistochemistry (IHC) have been used to identify the microorganism in the tissues (Fonseca, et al., 2006;Ooteman et al., 2006). Due to some limitations, culture of Leptospira is not always applicable (Obregón, et al., 2004;Rahim, 2005). Warthin-Starry (WS) silver staining has used to detect Leptospira and other spirochetes as a simple method in the tissues, thus, it could be applied in any laboratory (Fornazari, et al., 2012). The aim of this study was to nd out whether the Leptospiral infection is a serious zoonotic disease in Shiraz, Southern Iran, using developed nested PCR, MAT and WS silver staining methods.

Sample collection and preparation
Step one Total of 200 blood samples (113 male and 87 female) were collected from Nemazi Hospital, Shiraz, Iran. From which, 150 blood samples were taken from patients with a history and clinical symptoms suggestive of renal disorders, based on laboratory data including complete blood count (CBC), blood urea nitrogen (BUN), creatinine (Cr), total and direct bilirubin and urine analysis (WHO, 2003), the remaining 50 samples were belonged to healthy persons. The median age of the patients was 48.7 years (range = 13 -78 years). They were divided according to their age into three groups including ≤ 30, 31 -60, and ≥ 61. Blood samples were subjected to various hematological and biochemical tests including hemoglobin, platelet, red cell distribution width (RDW), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), hematocrit, red blood cell, serum creatinine and blood urea nitrogen. Some of the sera separated from blood samples were used for PCR and MAT tests.
Step two In this step, 20 and 10 nephrectomised kidney samples were respectively taken from patients following surgical operation and the rejected transplant kidneys which were apparently healthy. The apparently normal kidneys were considered as negative control. All the samples were subjected to the PCR assays, pathological examination including WS staining.
DNA extraction of serum and kidney samples 250 µl buffer I and buffer II containing RNase were initially added to the samples were pelleted. The mixture was then centrifuged at 10000 ×g for 10 min. A 550 µl saturated phenol was then added and pellets centrifuged at 10000 ×g for 5 min, the clear phase was then collected into new eppendorf tube. Previous stage was repeated twice to exclude cell debris. A 0.1 volume of 3 M sodium acetate was then added to the preparations, washed twice by absolute and 80 % ethanol, respectively. The tubes were centrifuged at 12000 ×g for 15 min., the nal pellet was dried at room temperature and re-suspended in 30 µl TE buffer, kept at -20 °C until further use.
Conventional and nested PCR to amplify 16s rRNA gene in the samples A conventional PCR assay was used to differentiate pathogenic and saprophyte species of Leptospira using separate speci c primers ( Table 1). The PCR was conducted using a 25 µl reaction mixture consisted of 2.5 µl of 10× PCR buffer, 1 µl of 10 mM dNTP mix, 1.5 µl of 25 mM MgCl 2 , 1.0 U of Taq DNA polymerase, 1 µl (25 pmol) of each primer and 50 ng of template DNA. Particular region of 16s rRNA gene of Leptospira spp was selected to design the primers (Table 1). All the pathogenic and non-pathogenic species of Leptospira were identi ed using a species-speci c nested PCR assay. The second ampli cation (nested-2) was ampli ed a 289 bp region within the 525 bp sequence from 16s rRNA gene. 1.5 µl of DNA was used to amplify both primary and secondary PCR ampli cation reactions in both reactions. The reaction was performed in a total volume of 25 µl of reaction mixture containing 1 mmol/L MgCl 2 , 1mmol/ml Deoxyribonucleotide triphosphate (dNTP) mixture (Sinagen, Iran) and 5 U/µl of Taq polymerase for both nested PCR reactions. The PCR reaction was conducted in a volume of 25 uL. A cycle was started with initial denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 60 °C for 2 min, extension at 72 °C for 1.5 min and a nal extension at 72 °C for 15 min. The conditions for secondary reaction were the same except for the annealing at 59.5 °C for 1 min, extension time for 1 min and nal extension time for 10 min. The ampli ed DNA fragments were run on 2% agarose gels electrophoresis stained with Safe mode staining and visualized on an ultraviolet transilluminator. Hauser USA) was used to adjust the organism to 2 × 10 8 leptospires/mL. The leptospiral standard antigen was prepared from six reference strains of Leptospira interrogans including Hardjo, Pomona, Icterohaemorrhagiae, Grippotyphosa, Canicola and Ballum. Serum samples were then serially diluted in phosphate buffer solution (PBS) in a microtiter plate (Greiner Bio-One, Kremsmünster, Austria). Recent infection is con rmed by very high antibody titers (≥ 400), but paired serum titers produce more reliable prognostic information. A current leptospirosis infection is suggested by a 4-fold rise in titer, or a seroconversion to ≥ 400. Numerous low-level titers most likely to indicate vaccination reactions, however this test should not be used to monitor protective vaccination status.

WS staining method
The staining method was performed using the WS stain kit (Asia Chem. Co., Tehran, Iran) as was described by the manufacturer. Brie y, the kidney sections were depara nised and rehydrated using milli-Q distilled water followed by immersing in a 1% silver nitrate solution incubated at 43 °C for 30 min. The sections were subsequently rinsed in distilled water and ooded in a fresh developer solution until turn to light brownish yellow, washed in hot water (56 °C) rinsed in distilled water, dehydrated, clean and mounted. The stain sections were nally examined under light microscopy (Mag. X 1000).

Statistical analysis
Spearman's rho correlation coe cient test was applied to assess the correlation between variables and chi square test (or Fisher's exact test) was used to analyze data using SPSS (version 18.0 for Windows, SPSS, Inc., Chicago, IL, USA) software. P values below 0.05 were considered as signi cant.

Nested PCR analysis of clinical samples in suspected cases
The nested PCR assay was con rmed that 30 out of 150 samples (20 %) were infected to the DNA of pathogenic species of Leptospira (Table 2). Seventeen out of 30 (56.7 %) positive patients by PCR were men. The ages of 31 to 60 years were the most affected age groups, though this was not statistically signi cant between men and women (P = 0.153) ( Table 2 and 3). The pathogenic species of Leptospira were differentiated according to the size of DNA amplicon (Figure 1). To develop a sensitive and speci c PCR assay for detection of all pathogenic and nonpathogenic Leptospira spp., two pair of primers were employed (Table 1). These primers were designed based on a particular region of 16S rRNA gene of Leptospira spp (Fornazari et al., 2012). According to the clinical pathological ndings, 28.5 % of patients showed anemia. The mean level of serum creatinine and blood urea nitrogen of positive patients were 3.6 and 46.9 mg/dl, respectively (Table 3). All the negative samples collected from healthy persons were shown negative PCR. The ratio of hemoglobin in the positive PCR cases were signi cantly lower than the normal values (P < 0.001).
Moreover, RDW was higher in all positive PCR patients (P < 0.001). A marked anemia was shown in the pathogenic strains PCR positives (P < 0.001) (Table 4). Microcytic, normocytic and macrocytic anemia were respectively shown in 56.7 %, 33.3 % and 10 % of the anemic patients. 60 % and 40 % of the patients were respectively males and females with the average of 40 years old. The incorporated kidney lesions were hyperemia, petechial hemorrhage, urinary cysts, shrinkage, pathological congestion, hydronephrosis and paleness, change in color and consistency and blood accumulation in the pelvis ( Table 5). Three of the 20 kidney samples of patients using nested PCR assay were positive for the pathogenic species of Leptospira (15%) (Figure 1) and all 10 samples of control group were negative. Positive samples were belonged to the male patients over 40 years old. No association was observed between kidney lesions and presence of leptospiral DNA. The Leptospira pathogenic species were con rmed in all the nested PCR positive kidney samples, however, no evidence of infection with the saprophytic species was observed.  Table 6 and 7. According to these results, positive titers were reported against L. Grippotyphosa (13 samples

Discussion
Leptospirosis is a widespread zoonotic infection worldwide, especially in the tropical and subtropical regions with the warm and humid climate which make favorite conditions for the survival of microorganism. Based on the information given by WHO, the infection is considered as a neglected disease with the estimation median of 1.5 cases per 100,000 each year in the endemic areas (WHO, 2003;Lau et al., 2012). Similar to other regions, the incidence of human leptospirosis is not well documented in the Middle East. Besides, the underestimating incidence, the complexity of diagnosis of infection is problematic (Murdoch et al, 2004). Little information is currently available on the occurrence of infection in human, in south of Iran. The present study was the rst molecular, serological and pathological work focused on the human leptospirosis in our country. Surprisingly, the disease was con rmed despite of the dry and hot climate summer with average temperature of 28.1 °C (19.3 to 37.0 °C), and cold winter with the average temperature of 6.0 °C (-0.3 to 12.4 °C) in this geographical area. The recently developed nested PCR, MAT methods and WS silver staining were employed. This study was also showed a numerical relation of the most affected age group which was between the ages of 31 to 60 years in both genders. A comparison between the occurrences of infection in different age groups in the former studies showed a higher prevalence in the patients above 50 years old (Honarmand, 2009). A higher rate of occurrence in Iran was also con rmed in the ages above 40 years old (Esmaeili et al., 2009). Nested PCR con rmed that 30 (20 %) of 150 serum samples infected to the Leptospira infections. Additionally, the conventional PCR assay was approved that all positive cases had a pathogenic Leptospira species. The sensitivity of nested PCR to identify Leptospira spp. was approved by Djadid et al. (2009) andAhmed et al. (2012). The primers were designed based on 16S rDNA gene sequence, which is able to identify all pathogenic and non-pathogenic species of Leptospira, to detect 1 to 2 pg of DNA in a sample (Djadid et al. 2009). The ratio of hemoglobin in the positive cases were signi cantly lower than the normal values (P = 0.05). Moreover, RDW was higher in all positive PCR patients (P < 0.001). RDW is an approved index to assess anemia. Increase in the proportion of large, young cells with older and smaller red blood cells should directly associate with anemia. In the immune-mediated hemolytic anemia there are often immature cells mixed with normal ones. The RDW should be particularly high with immune-mediated hemolytic anemia (IMHA) where there are often immature cells mixed with cells that are small due to losing pieces of their membranes (spherocytes). Unfortunately, the RDW does not always re ect the degree of anisocytosis seen on peripheral blood examination, which emphasizes the importance of smear evaluation (Stockham, and Scott, 2013). A marked anemia was shown in the pathogenic strains PCR positives patients (P < 0.001). Low serum creatinine, severe anemia and low hematocrit values were observed in acute form of leptospirosis (Reis et al., 2013). In our study, the incidence in the Northeast province of Iran were 27.29 % and 28 % in men and women, respectively (Sakhaee et al., 2010). MAT assay was employed to con rm leptospira in 400 samples, 194 (48.5 %) were found positive, of which, the highest and lowest titers were respectively recorded against L. gripotyphosa and L. hardjo. In addition, a higher prevalence of infection in women was noticeable (Ebrahimi et al., 2015). Occurrence of various leptospira Spp. in different climates of Iran is mainly associated with the presence of appropriate reservoir, sensitivity of hosts and methods of leptospira surveillances in our country. Survival of microorganism in wet areas made it endemic in the north of the country (Ko et al., 2009;Sakhaee and Abdollahpour, 2011). Here, we have also shown the occurrence of infection in the dried and semi-dried regions which con rmed that the epidemic is less dependent on the ambient humidity. The formalin-xed tissues was employed to apply on the WS staining. The effectiveness of WS for detecting the microorganism have been already approved by some previous studies (Sebastian et al., 2005;Léon et al., 2006;Ortega-Pacheco et al., 2008). However, the positive cases con rmed by WS technique, were fewer than PCR. The low e cacy of WS to detect Leptospira was possibly associated with low burden of microorganism in tissues. Un-speci ed staining of reticulin bers to mis-interpreting results of WS makes it problematic to properly differentiate the infection (Szeredi and Haake, 2006). In the current study, pyelonephritis was more prevalent than other lesions because the microorganism is a nephrophilic and can generate many renal lesions (Visith and Kearkiat 2005). White spotted lesions are repeatedly observed as an important signs of chronic leptospirosis, whilst, inspection of 329 healthy cattle's kidneys, showed that both Leptospira spp. and active infection caused by other bacteria are associated with the "white spotted kidneys" (Uzal et al. 2002a;Uzal et al. 2002b).

Conclusion
The clinical, serological and epidemiological ndings from our studies in Fars province, Iran were implied on the presence of pathogenic species in human. However, overall information on the dynamic of transmission of leptospira was suggesting an emerge focus on the occurrence of disease especially in the global travelers. The results of present study highlight the urgent need to achieve further details about the possible role of the bacteria to induce renal failure in various climate conditions, in Iran.

Declarations
Funding: Not applicable.
Competing Interests: The authors declare no nancial and non-nancial competing interests.

Ethic approval
These experiments were permitted by the members of state ethic committee, Shiraz Uni versity, Shiraz, Iran (IACUC no: 4687/63). It is con rming that informed consent was obtained from all subjects.
Availability of data: Data produced during this study are available from corresponding author upon reasonable request.

Figure 2
Warthin-Starry (WS) staining of kidney. Presence of liform, dark brown Leptospira (arrows) in a secretion in the lumens of cortical tubules.