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 Table of Contents  
Year : 2022  |  Volume : 24  |  Issue : 1  |  Page : 26-31

The blue 'solution': Evaluation of methylene blue as a screening test for the diagnosis of significant bacteriuria

1 Department of Rural Service, St. John's Medical College, Bengaluru, Karnataka, India
2 Department of Microbiology, St. John's Medical College, Bengaluru, Karnataka, India

Date of Submission22-Apr-2022
Date of Acceptance10-Jun-2022
Date of Web Publication11-Jul-2022

Correspondence Address:
Anna Rachel Menezes
St. Ann's Hospital, Elathagiri Road, Elathagiri, Krishnagiri - 635 108, Tamil Nadu
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jacm.jacm_7_22

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BACKGROUND: In the year 2014, Nnaemeka and Iyioku in Nigeria developed the methylene blue screening test and advocated for further study regarding the evaluation and usage of the test in a low-resource setting. In this study, we aimed to establish a clear cut-off absorbance for a good predictive value, evaluate the sensitivity and specificity of the test, compare the results to available screening methods and determine the applicability of the test in screening for bacteriuria that does not adhere to Kass criteria and hence thoroughly evaluate the test for external validation before use in low-resource settings.
MATERIALS AND METHODS: In this study, a total of 217 urine samples were run through the modified methylene blue test and culture, i.e., the gold standard for diagnosis of significant bacteriuria; using the results, a cut-off was derived using receiver operating characteristic curve analysis. Statistical analysis was then performed using the cut-off. The minimum colony count was determined for the given cut-off to predict whether the test can be used in different settings of bacteriuria. The above results were then compared to available screening tests based on a thorough literature search on PubMed.
RESULTS: Using 0.36 as a cut-off absorbance value, the calculated sensitivity and specificity of the test were 83.5% and 92.9% with a positive predictive value of 90% and a negative predictive value of 87.40% according to the current prevalence of urinary tract infection in South India. The P value of the above result determined using the Chi-square test was <0.05 and hence the results were deemed to be statistically significant.
CONCLUSION: The test could precisely diagnose bacteriuria with a colony count of >105 colony-forming units/mL and had comparable sensitivity and specificity to a combination of leucocyte esterase and nitrite tests which are popularly used as screening methods.

Keywords: Methylene blue test, screening, urinary tract infection, urine culture

How to cite this article:
Menezes AR, Mishra B. The blue 'solution': Evaluation of methylene blue as a screening test for the diagnosis of significant bacteriuria. J Acad Clin Microbiol 2022;24:26-31

How to cite this URL:
Menezes AR, Mishra B. The blue 'solution': Evaluation of methylene blue as a screening test for the diagnosis of significant bacteriuria. J Acad Clin Microbiol [serial online] 2022 [cited 2023 Jun 5];24:26-31. Available from: https://www.jacmjournal.org/text.asp?2022/24/1/26/350321

  Introduction Top

Urinary tract infection (UTI) is an umbrella term, implying an infection in any part of the urinary tract ranging from the urethra to the kidneys.[1] It remains the most common bacterial infection in the human population, having a higher prevalence in developing countries.[2],[3]

The symptoms of UTI include burning micturition, fever and flank pain, although bacteriuria without symptoms is prevalent in pregnant women.[4] UTIs are known to increase the risk of pyelonephritis, premature delivery and foetal mortality in pregnancy and impairment of renal function and end-stage renal disease in paediatric patients.[3]

A large number of samples are sent to microbiology laboratories for culture which is the gold standard for the diagnosis of UTI.[5] Of which, almost 60% of samples do not show any growth in culture media, wasting important laboratory resources.[6] The samples also require a long turnaround time of 48 h.[5] The misdiagnosis of UTI due to contaminated urine cultures also leads to the indiscriminate use of antibiotics promoting antimicrobial resistance.[7],[8] Furthermore, diagnosis and treatment of UTIs cost the global economy a large amount of almost US $6 billion.[9] Thus, it becomes indispensable to have a rapid screening method to screen urine samples for significant bacteriuria.[10]

Literature on methods currently used for the screening of urine samples prior to culture, is as follows:

The positive nitrite and/or trace leucocyte test was analysed by Shimoni et al., 2017, and the sensitivity was found to be 96.9% which correlated with a similar study done by Schroeder et al., 2015.[11],[12] Schroeder also analysed urine for pyuria and found the sensitivity to be 96% which is in close agreement with a study done by Hoberman et al., 1996.[12],[13]

The cost-effectiveness of the dipstick test was studied by Shajari et al., 2009, and the test was found to have an expense of 0.092$ per patient, which is fairly high in a community setting.[14] The urine dipstick test also produces false-negative results in scenarios where non-nitrite-producing species of bacteria such as Enterococcus or Staphylococcus are involved or when patients have been treated with certain drugs.[5],[15]

In 2015, Gill et al. studied the effectiveness of detection of bacterial adenosine triphosphate in urine and concluded that the test had no useful diagnostic role.[16]

Newer methods including mass spectrometry, fluorescent in situ hybridisation and polymerase chain reaction, while being highly sensitive, specific and time-saving, demand a heavy healthcare expenditure and also require a high level of technological development.[17]

In 2014, Nnaemeka and Iyioku devised a cost- and time-effective method for screening samples for significant bacteriuria.[6] This method used easily available and inexpensive materials such as methylene blue and saline. Twenty microlitres of methylene blue was added to 10 mL of each of the urine samples and their absorbance was read at 540-nm wavelength. A standard was prepared by adding the same amount of methylene blue to normal saline. The absorbance of the standard was considered a cut-off value. If urine samples had an absorbance greater than that of the cut-off they were labeled as positive for significant bacteriuria, those lower than or equal to the cut-off were labeled as negative. The sensitivity of the test was calculated to be 95% and the specificity of the test was calculated to be 97.17%, by the inventor of the study as described in his article.[6]

A similar study was undertaken by Mokhtar and Roshdy in the year 2017. They observed that the specificity and sensitivity of the test to correctly detect significant bacteriuria were 89% and 77%, respectively.[5]

Although both the studies adequately determined the sensitivity and specificity of the test they did not establish an appropriate cut-off the value that could be used universally, there were also no comments on whether the test could be used to screen for bacteriuria that does not adhere to the Kass criteria of 105 colony-forming units (CFU)/mL which include samples from pregnant or sexually active females with pyuria where a criterion of 102 is used and samples used to diagnose cystitis or pyelonephritis in the elderly where criteria of 103 and 104 are used, respectively.[18],[19],[20]

In this study, we focused on evaluating the sensitivity and specificity of the modified methylene blue test using urine culture as a standard. Using receiver operating characteristic curve (ROC) analysis, we established an appropriate cut-off value and hence evaluated the predictability of the methylene blue test. We also studied the relationship of colony count with the results of the modified methylene blue test and endeavoured to establish the number of colonies required for the modified methylene blue test to show a positive result to evaluate the usage of the test in different settings. Finally, a thorough PubMed search was done to review the available literature on current trends of contemporaneous screening methods and to compare the results with the methylene blue test.

  Materials and Methods Top

Ethical consideration

Ethical clearance was sought and obtained from the Institutional Ethics Committee reference number: 123/2018.

Study site and duration

The study was conducted in the Department of Microbiology and the Department of Biochemistry during the course of two months at a tertiary and referral hospital in South India.

Source of data

In this hospital-based study, urine samples collected routinely at a tertiary care hospital and sent to the microbiology laboratory for culture and sensitivity testing were used.

Inclusion criteria

All samples that were routinely collected from the tertiary care hospital and sent to the microbiology laboratory for routine culture and sensitivity testing were included in the study.

Exclusion criteria

(1) Samples that were leaking from the container, (2) samples that were delayed for more than two hours without refrigeration for processing and (3) cloudy urine samples from which 10 mL of urine could not be easily pipetted were excluded from the study.

Sample collection

Patients were instructed by hospital staff to wash both hands and genitals with soap and water to ensure the aseptic method of collection of midstream urine. The amount of urine collected should be a minimum of 15 ml. Catheterised urine was collected from the port after appropriate cleaning.

The urine samples were collected in 40-mL sterile blue-capped containers with identification numbers and transported using sterile container boxes from each ward to the microbiology laboratory within 30 min. The samples were immediately refrigerated at the microbiology laboratory and sampled within 12 h using the techniques mentioned below:

After culture, the samples were re-refrigerated for 48 h before being discarded.

Sample analysis

Semi-quantitative urine culture

All 217 samples were cultured on 5% sheep blood agar and cysteine–lactose–electrolyte-deficient (CLED) agar within two hours of sample collection. The plates were cultured using inoculation by a calibrated loop of 2 mm or 0.005 ml. The modified Mayo technique was used to streak the culture plates. The plates were then aerobically incubated at a temperature of 37°C and inspected after 18–24 h.

Modified methylene blue test

The technique for the procedure demonstrated by Nnaemeka and Iyoku is as follows:[6]

Preparation of reagent

The reagent was prepared by adding 5 g of methylene blue powder obtained from HiMedia Laboratories Pvt. Ltd., Maharashtra, India, to 1 l of distilled water, and the solution was mixed well. The same solution was used throughout the study [Figure 1] and [Figure 2].
Figure 1: Apparatus required for the methylene blue test

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Figure 2: Methylene blue reagent

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Preparation of standard

The standard was prepared by adding 20 μl of methylene blue reagent to 10 ml of normal saline of concentration 0.9%. It was allowed to stand at room temperature for five minutes [Figure 3].
Figure 3: Preparation of the standard and samples labeled 1 and 2

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Preparation of test samples

Ten millilitres of each of the 216 urine samples were added individually to a 15-ml test tube, and 20 μl of the prepared methylene blue reagent was then added to this using an adjustable micropipette. The mixture was then allowed to remain a room temperature for a period of five minutes.

Reading the samples

1.3000 μl of the standard was pipetted out of the test tube and transferred into a 5-ml cuvette. The absorbance of the mixture was then read on a SUNTRONICS INDIA® digital photo colorimeter St-211 at 540-nm wavelength against a water blank. Three such readings were taken and the average value was deemed as the standard.

The O. D of the standard against water blank at 540 nm was: 0.26.

Two thousand five hundred microlitres was pipetted out from each of the test tubes containing the urine–methylene blue mixture and transferred individually to a 5-ml cuvette. The absorbance of the urine–methylene blue mixture was also read on a SUNTRONICS INDIA® digital photo colorimeter St-211 at 540 nm wavelength against a water blank. The reading of each sample was noted down.

Labelling the samples

All samples that had an absorbance value of <0.36, which was the cut-off value determined using ROC analysis, were labeled as positive samples and those >0.36 were labeled as negative samples.

Statistical analysis

The data were analysed using a Chi-square test and simple descriptive statistics on SPSS 26 (IBM: Statistical Package for Social Sciences (SPSS), Chicago, USA). P value was considered statistically significant at a 95% confidence interval. The tests were two-tailed. P < 0.05 was considered to be statistically significant.

  Results Top

Urine culture

Out of the 217 samples that were cultured in both 5% sheep blood agar and CLED, 97 (44.70%) were found to have CFU per ml <105, i.e., significant bacteriuria.[19] Thirty-six samples had 104–105 (16.58%) CFU per ml.

Modified methylene blue test

As shown in [Graph 1], the area under the curve was 0.885 with a standard error of 0.015, showing that the test will have a high sensitivity and specificity with a cut-off of 0.36.

Using 0.36 as the cut-off value

On using 0.36 as the cut-off value, 81 out of the 97 samples showed true positivity with the methylene blue test, and 16 showed false negativity. Out of the 120 samples that were negative on urine culture, 111 were tested true negative and 9 were tested false positive.

Using the above data

Sensitivity was calculated to be 83.50% and specificity was calculated to be 92.9%.

According to [Table 1], the Chi-square test statistic is 127.6593 and the P value is 0.0000 (P < 0.05) which is statistically significant.
Table 1: Statistical association between modified methylene blue test and urine culture results, using cut-off value as 0.36

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Calculating the minimum colony count for the methylene blue test to show positivity

In [Table 2], it is seen that the highest rate of positivity was seen with samples having a colony count of ≥105 CFU/ml, and hence according to our study, the modified methylene blue test correctly determines samples having a colony count of ≥105 CFU/ml. This implies that urine samples having bacteriuria below Kass criteria cannot be correctly identified using the cut-off.
Table 2: Results of modified methylene blue test concerning colony count in colony-forming unit/ml

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Comparison of the methylene blue tests to available screening tests

A thorough search on PubMed was performed to collate the sensitivity and the specificity of the methylene blue test to available screening tests, using keywords such as 'Urinary Tract Infection', 'Screening', 'Urine Culture' and the names of the specific screening tests.

The results obtained are visualised using [Table 3]. It is seen that the sensitivity and specificity of the modified methylene blue test are comparable to that of a combination of nitrate and leucocyte esterase test.
Table 3: Comparison of sensitivity and specificity of available screening tests

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  Discussion Top

The diagnosis of UTI becomes paramount as the symptoms may vary from asymptomatic bacteriuria to sepsis.[4] The current gold standard is urine culture.[32] Our study revealed that only 133/217 (61.28%) samples were culture positive, in a tertiary and referral hospital setting. This is higher than the usual rate of around 20%–40% of cultured samples showing growth.[5],[6] The above was attributed to the study being conducted in a tertiary referral hospital setting where a large number of patients are severely ill, and additional possible reasons for increased observance in urine culture positivity include an improper method of urine collection and transport.[33]

The few available screening tests have either a low sensitivity or a high expenditure.[9],[29] In our study, we studied the effectiveness of the modified methylene blue test to detect significant bacteriuria and found the sensitivity and specificity to be in the same range as the combined dipstick test, being 83.5% and 92.9%, respectively.

The modified methylene blue test detected a total of 81 (90%) positive (absorbance above the cut-off value) and 111 (87.4%) negative (absorbance below the cut-off value) which is following the study conducted by Nnaemeka and Iyioku in Nigeria, who introduced this method in 2014.[6] We also studied that a minimum colony count of 105 was required for the modified methylene blue test to show true positivity; this was also shown by Mokhtar and Roshdy in a study conducted at Cairo University.[5]

Therefore, the advantages of the modified methylene blue test were its short turnaround time, use of easily available instruments such as the spectrophotometer, lack of need for trained laboratory personnel to perform the test and its inexpensive nature.

  Conclusions Top

From this study, we concluded that the modified methylene blue test not only had good sensitivity and high specificity but also could be easily performed in low-resource settings. An appropriate cut-off for absorbance of the modified methylene blue test would be 0.36. The methylene blue test can correctly identify urine samples that have grown more than 105 CFU/mL and hence can be effectively used to prevent wastage in resource-limited settings.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

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  [Figure 1], [Figure 2], [Figure 3]

  [Table 1], [Table 2], [Table 3]


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