Urine cytological examination: an appropriate method that can be used to detect a wide range of urinary abnormalities [version 1; peer review: 1 approved, 1 approved with reservations]

Background: Urine cytology is a method that can be used for the primary detection of urothelial carcinoma, as well as other diseases related to the urinary system, including hematuria and infectious agents. In this study we aimed at investigating urine abnormalities among Sudanese patients attending Omdurman teaching hospital. Methods: A descriptive cross-sectional study was conducted from November 2016 to October 2017. A voided urine samples were collected and stained using Papanicolaou stain. Results: A total of 1238 urine samples were meticulously examined, 832 (67.2%) from males (mean age 41.7±12.67), and 406 (32.8%) from females (mean age 43.8±10.94). 147 (11.9%) patients had an underlying medical condition, either AIDs, diabetes mellitus type 2 or historical renal transplantation. Hematuria was more frequent amongst males than females, 100 (68.9%) and 45 (31.1%), respectively. Open Peer Review


Introduction
Urine cytology is a safe and inexpensive method to diagnose urinary tract abnormalities. It is similarly used for the detection of primary and recurrent urothelial carcinoma 1,2 ; as well as in many other diseases related to the urinary system such as urinary schistosomiasis 3 and bacterial infections 4 . Urine cytology has variable sensitivity and specificity depending on the sample collection method and tumor grade in the case of malignancy 5-7 . Although the determination of cell morphology through microscopy remains the gold standard 8 , false positive and negative results have been reported. The false-positive results from urine cytology may be attributed to the presence of viral infections, such as polyomavirus. False negative results have been linked to the sampling method used, the number of the samples obtained from the patient and the volume of the urine being processed 1,6-10 . Urothelial carcinoma is one of the most commonly reported cancers 11 . The major clinical concern for the management of bladder cancer patients is that most of the patients relapse 12,13 . Therefore, regular follow-up is required for patients who are at risk of urothelial carcinoma 14 . Despite many attempts to develop tests with higher sensitivity and specificity, cytology remains one of the best ways to diagnose a variety of bladder lesions 15 .
Hematuria is one of the common indications for urine cytology examination. It is also known to be caused by an underlying malignancy in 5% to 10% of patients 16 . Urine cytology is also used for the detection of abnormalities related to diseases like schistosomiasis 3 , candidiasis 17 , polyomavirus (BK virus (BKV)) infection 18,19 , Human Papillomavirus (HPV) 20,21 and cryptococcosis 22 . In this study, we aimed at investigating the frequency of urine abnormalities among Sudanese patients attending the outpatient clinic at Omdurman Teaching Hospital, Khartoum, Sudan using urine cytology.

Methods
A descriptive cross-sectional study conducted from November 2016 to April 2017, an organized mass screening programme for urine cytology was introduced during the study period. The target screening population consists of the entire patients attending the outpatient's clinic and agreed to participate in the study. A total of 1238 Patients were recruited from the outpatient clinic at Omdurman Teaching Hospital in Khartoum state, Sudan. All the patients attending the outpatients clinic were assessed by our research team and we discussed with all the patients the objective of our research. After obtaining written informed consent from the patients whom were willing to participate our research team gathered the patients information and demographic data including name, age, and any clinical signs or symptoms using a questionnaire, the process of filling and collecting the patients clinical data were conducted by our research team and it took place in the patients waiting area in the clinic, focusing on patients with HIV, diabetes mellitus and transplant recipients, and any patients with other conditions mentioned in the questionnaire.

Sample collection
Early in the morning, first pass urine sample was rejected as the cells may be abnormal, with features such as enlarged nuclei and washing out of the chromatin, features that mimic malignant cells; this is due to the pH of the urine 23-25 . Accordingly, the patients were asked if they passed urine before and if they drank water? Then each patient was provided with a sterile screw cap urine container and asked to pass some urine into the toilet at first, then without stopping the flow of the urine catch some in the provided container.

Sample processing
The entire volume of the urine samples were centrifuged (using Hettich® EBA 20 centrifuge, Germany) for five minutes at 3000rpm then the supernatant was discarded leaving the sediment, this was followed the addition of suspending media (74 ml saline, 6 ml Glacial acetic acid and 20 ml of 100% ethanol) to the sediment. The quantity of the suspending media was proportional to the quantity of the obtained sediment this is done by measuring the sediment quantity using automated pipette and the same quantity of the sediment was added the suspending media (after centrifugation we measured the quantity with a pipette). It was left for 30 minutes, followed by centrifugation for 5 mins at 3000 rpm. The supernatant was discarded and 1% acid alcohol (1 ml of Hydrochloric acid added to 99 ml of 70% alcohol) was added to the sediment and left for 30 minutes, followed by centrifugation at 3000 rpm for 10 mins, and discarding of the supernatant again. The resulting sediment was then used for smears by spreading the entire obtained volume on a microscopical slide, and then the slide were left to concentrate. Then the smears were fixed in 95% ethyl alcohol (V/V) for 15 mins.
The smears were stained using Papanicolaou staining procedures as follow: the smears were immersed in a descending gradient of alcohol from 80%, 70% to 50% (V/V) and then immersed the smear in tap water for 2 mins. Then the smears were stained using Harris Hematoxylin for 2 mins, after that the smears were washed in tap water and differentiated in 1% acid alcohol for seconds. This was followed by bluing by leaving it in a bath of running water for 10 mins, then immersed in 80% and 95% alcohol and stained with Orange G solution (O.G.6) for 2 mins. This is followed by washing in 95% alcohol, and then staining with Eosin Azure (EA.50) for 4 mins and washed again in 95% alcohol. Then rinsed quickly in 100% alcohol and blot using a sterile filter paper to dry. Clear in Xylene and mounting in distyrene (a polystyrene), a plasticiser (tricresyl phosphate), and xylene (D.P.X) (Catalog No. 100579; sigma -Aldrich, USA) 14 .
For reporting the cytological smears, two cytopathologist (EES, MAM) examined the entire slides blindly using Olympus light microscope (Model No. CX21FS1, Olympus, Tokyo, Japan). The cytological diagnosis was grouped into 5 categories according to The Paris System for Reporting Urinary Cytology (PSRUC) 23 , categories included nondiagnostic/ unsatisfactory, negative for high-grade urothelial carcinoma (NHGUC), atypical urothelial cells, suspicious for high-grade urothelial carcinoma (SHGUC), high-grade urothelial carcinoma (HGUC) and Low-grade urothelial neoplasm (LGUN). The presence of koilocytosis, decoy cells, and pseudo-hyphae, as well as detecting the variable sizes of cells with clear hollow and narrow base with single budge were used as a diagnostic remark for the presence of other urinary abnormalities such as HPV and BKV. Urine cytology results obtained by the cytologists were sent to the physician who made the final decision on the patients' diagnosis for proper patients management according to their findings of the cytological smears.

Statistical analysis
Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS. Version 16). Chi-square test was used to compare urine cytology results with age, gender and to test the odds ratios of patients showing abnormalities in relation to their medical condition (A p value of <0.05 was considered to be statistically significant).

Analysis of urine cytology results based on patients' age group
Regarding the relationship between age and cytological findings, our study showed that candida infection, Schistosomiasis, Polyoma virus and HPV were highest among the age group 41 -60 years; while Cryptococcus infection was highest among 21 -40 years old patients.
A representative microscopic result of urine cytology diagnosis is shown in Figure 1, and the distribution of gender, presence of hematuria, medical condition and urine  cytology diagnosis based on age groups is described in Table 2.
Gender risk analysis for having urine abnormalities Of the 1238 patients, a relative risk estimation was conducted for both male and female with any of abnormal cytological finding and the results showed a significant proportion of females to be infected with cryptococcosis and HPV; p value = 0.001, odds ratio was 0.  Table 4.

Discussion
Urine cytology is considered a noninvasive, cost effective technique that is able to diagnose a wide range of abnormalities including infections, atypical cellular changes and malignancy 26,27 . However, in this study, we investigated a varied array of urine abnormalities among Sudanese patients attending the outpatient clinic at Omdurman Teaching Hospital, Khartoum-Sudan using urine cytology.
In this study, we observed that urine cytology could easily differentiate benign from malignant cells showing high specificity for detecting HGUC; this result agrees with previous reports 8,28-30 . HGUC are aneuploid and are detected with high sensitivity, as compared to atypical urothelial cells 29-31 . The cytological features of HGUC identified in this study showed that the smears exhibited high cellularity and dehiscence cellular pattern. Malignant cells were larger than normal cells and showed pleomorphic nuclei with prominent nucleoli. Also, the cytoplasm ranged from homogenous to scant vacuolated one. Depending on these features, previous studies have indicated that detection of malignant cells using urine cytology is the gold-standard method for diagnosing urothelial cancer. 2,29,32 .
In this report we are also able to identify Candida spp with great confidence by the presence of the budding yeast and pseudohyphae which is considered as the cytomorphological characteristic of this fungus, however if the organism was not identified, cytological changes such as stain anomaly, nuclear degeneration and bacterial background may pinpoint the presence of candida.
Cryptococcus infection was detected in 2 out of 13 HIV patients; 0 out of 69 DM2 patients and 2 out of 65 renal transplant recipients. The organism was correctly identified by the presence of a semitransparent fungal organism with double contours in an inflammatory background 22,31,33 .
Polyoma virus (BKV) was detected in 0 out of 13 (0%) HIV positive patients; 2 out of 69 (2.89%) DM2 patients and 6 out of 65 (9.2%) renal transplant recipients. The key feature for the existence of polyomavirus is depending on the presence of the unique cytomorphological features, i.e., presence of the typical decoy cells, the nuclei of this cell appeared as uniform with washed chromatin 34-42 . The results obtained by this study demonstrated that urine cytology can be used as a simple monitoring test for the renal transplant recipient for the presence of polyoma virus, since polyoma virus infection in renal transplant recipient is considered as one of the factors that can lead to graft rejection 42-48 . Our results are in concordance to that conducted by Kapila and his associates; they investigated the diagnostic efficacy of urine cytology in detecting Polyoma virus among renal transplant recipients comparing urine cytology with molecular diagnosis and their results showed that urine cytology can be used as an easy tool for monitoring the patients 49 . Furthermore, our results showed that no polyoma virus was been detected among HIV patients, which is in contrast to the findings of Boldorini and his associates as they screened 78 patients with HIV for the presence of polyoma virus using urine cytological examination and molecular and immunohistochemistry, there results demonstrated that out of 78 patients, only 17 patients were positive for Polyoma virus using cytology 50 .
Furthermore, Human papilloma virus was been detected among 9 out of 1238 (0.7%) of whom 8 were females and one was a male; the results obtained here should be evaluated with care especially in the settings of female individuals as it is very difficult if not impossible to differentiate whether these koilocytic cells originated from the urinary tract or they belong to the uterine cervix and therefore in such circumstances we highly recommend that these females should go for pap smear to locate the exact site of HPV infections.

Conclusion
Urine cytology is a safe, noninvasive, and reliable diagnostic tool for identifying viral cytopathic effects in urothelial cells, and deserves more widespread use in the monitoring of patients, especially those with renal transplants. It now seems that urine cytology needs to be implemented in clinical settings for the benefit of patients and urologists. Polyoma virus infection detection among transplant patients helps urologists to identify and manage renal graft rejection early which is of invaluable benefit to patients, physicians and economically. Due to the risk of the presence of polyoma virus among renal transplant recipient and based on the fact that 50% of the cases with Polyoma virus was associated with the graft rejection we highly recommended to use urine cytology as an early screening tool for routine checks among renal transplant patients. Furthermore, Pap staining was considered as an excellent cytological stain that provided excellent morphological quality for the visualization of the nuclear changes.

Ethics approval and consent to participate
The study was approved by the Faculty of Medical Laboratory Sciences Research Ethics Committee -University of Khartoum, Sudan (Nov/2016/102). Written informed consent was obtained from each participant prior to participation. This is a well conducted study and a well written manuscript; easy to read, but with scattered linguistic errors.

Data availability
I have a few comments: Your study population are patients referred to the out patient clinic, I presume. I see that only a minority have a condition, but they must have had some symptom(s) or cause for their referral. Even if it turned out that there was no specific condition, you should state the cause for referral.
You have 3 cases with a cytological diagnosis of low grade (atypical) urothelial cells. The Paris system is only concerned about high grade atypia. In which category did you put these three cases?
Under discussion: second last paragraph: ...conducted by Kapila and her associates. She is a woman (and a friend of mine).
Under conclusions, you could add a sentence or two about the value of urinary cytology in the diagnosis of infectious conditions as you so nicely demonstrated with cryptococcus and Schistosomiasis. This would be most valuable in areas where tropic/subtropic infections of the urinary tract are widespread.

Is the study design appropriate and is the work technically sound? Yes
Are sufficient details of methods and analysis provided to allow replication by others?
detecting HGUC'. To determine specificity we have to compare the results with pathologic examination. If there is a cytology-pathology correlation in this study, it is better to mention in "method and result" sections with proper tables.

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound? Partly

If applicable, is the statistical analysis and its interpretation appropriate? Yes
Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Partly
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