Evaluation of darkground microscopy as a rapid diagnosis procedure in leptospirosis
Background & objectives: Although darkground microscopy (DGM) is the method employed for examining the growth of leptospires in culture, it is not useful for diagnosis when used directly on body fluids. However, it continues to be used as a method for diagnosing leptospirosis in many centres in India. The present study was undertaken to evaluate the diagnostic accuracy of DGM by comparing it with established tests like culture, microscopic agglutination test (MAT), IgM ELISA and Lepto– Dipstick.
Methods: A total of 170 patients clinically suspected to have leptospirosis were included in the study. The gold standard for diagnosis was positive blood culture, or seroconversion, or a four-fold rise in titre in MAT. DGM was done on plasma after centrifugation at 1000 g and 3000 g and on serum. Indices of accuracy including sensitivity, specificity, predictive values and kappa value of agreement with the gold standard diagnostic criteria were calculated.
Results: DGM had a sensitivity of 40.2 per cent, specificity of 61.5 per cent, a positive predictive value of 55.2 per cent and a negative predictive value of 46.6 per cent. If had agreement with the gold standard in only 50 per cent cases the kappa value being 0.017141.
Interpretation & conclusion: DGM has low indices of accuracy. The results obtained in patients who met the gold standard criteria for diagnosis and those who did not were identical indicating that the test results are not determined by the presence of leptospiral infection in the patient. DGM, therefore, is not recommended as a sole diagnostic procedure for early diagnosis of leptospirosis.
Key words Andaman – darkground microscopy – leptospirosis
Received November 3, 2000
Leptospirosis is emerging as an important public health problem in the developing countries and is often an aftermath of natural disasters1. The disease has a wide range of manifestations and it is difficult to differentiate it from many other infectious diseases purely on clinical grounds. Hence the diagnosis depends on laboratory evidence of leptospiral infection. Leptospires are usually present in the blood during the first week of illness and are excreted in the urine from the second week onwards up to a month or even longer2.
Demonstration and isolation of leptospires from clinical material is definite proof of infection. Isolation needs an elaborate laboratory set up, and even under ideal laboratory conditions the success rate could be low. Demonstration of organisms by darkground microscopy (DGM) appears to be a simple alternative. However, the number of leptospires circulating in the blood may be too low to allow direct detection under the microscopy3. In addition, serum proteins and fibrin strands may resemble leptospira under darkground illumination leading to false positive results.
With increasing awareness of the disease, many investigators have started working on leptospirosis. Due to non-availability or on account of the expense of procedures many investigators including those working in several well -established laboratories have been using DGM as a reliable method of diagnosis 4-7. This could result in overestimating the incidence of leptospirosis in several parts of the country. We, therefore, considered it significant to undertake this study to evaluate the accuracy of DGM as a diagnostic tool.
Material & Methods
A total of 170 patients clinically suspected to have leptospirosis, who were either attending the OPD or admitted to the G.B. Pant Hospital, Port Blair or Primary Health Centre, Manglutan, South Andaman during September 1998 to August 2000, were included in the study. The clinical criterion for suspicion of leptospirosis was the presence of acute onset of fever, headache and body aches associated with any of the following: (i) Cough, haemoptysis and breathlessness; (ii) jaundice; (iii) reduced urine output; (iv) any haemorrhagic tendencies including sub-conjunctival haemorrhage; and (v) severe calf muscle tenderness.
Five to six ml of blood was drawn aseptically on the day of reporting. Half of the sample was transferred to a sterile tube containing 0.3 ml of I per cent sodium oxalate in 0.OIM phosphate buffer pH 7.4 and the other part to another tube without anti-coagulant. These tubes were transported to the laboratory within I to 2 h of collection of the samples and serum/plasma was separated. Follow up samples were collected 7-14 days after the collection of the first sample. Blood culture and DGM were performed on all the acute samples. MAT, IgM ELISA and Lepto-Dipstick were done on both the acute and follow up samples.
Blood culture was performed following standard procedure2. Briefly, two screw-capped test tubes containing 5 ml each of Ellinghausen, McCullough, Johnson and Harris (EMJH) medium (base: DIFCO, USA, enrichment prepared locally) was used for each patient. Two drops of blood were added to the first test tube and 3 drops to the second. The inoculated media were incubated at 28-30 deg C and examined weekly under darkground microscope (Olympus CH 40, Japan) for leptospires.
For the demonstration of leptospires in the specimen, differential centrifugation method was performed as described by Wolff8. Oxalated blood was centrifuged at 1000 g for 15 min. A thin film from one drop of plasma was examined under dark ground illumination. The negative plasma samples were centrifuged again at 3000 g for 30 min and reexamined in the same way. Similarly a drop of serum from the tube not containing the anti coagulant was also examined under darkground illumination. DGM was performed by two scientists (PV, TU), who had at least five years of experience in leptospira laboratory work. The tests were performed blindly without knowledge of the clinical status of the patients. The test was considered positive if both the observers agreed that they observed leptospira like motile structures.
IgM ELISA, Lepto Dipstick and MAT were performed with the serum samples employing standard procedures8-10. The ‘Gold Standard’ for diagnosis was (i) positive blood culture; or (ii) seroconversion from negative to positive with minimum titre of 1:100; or (iii) a four-fold rise in titre in MAT.
The positivity rates of DGM, IgM ELISA and Lepto-Dipstick were calculated in patients who fulfilled the `Gold Standard’ criteria for diagnosis and patients who did not. Sensitivity, specificity and predictive values of DGM as well as its agreement with `Gold Standard’ and kappa value were calculated. The agreement between the two observers and kappa value of agreement were also calculated.
To estimate the concentration of leptospires in the specimen necessary for successful detection under DGM, suspensions of leptospires from culture were made in human blood at different concentrations (1-4x 10^sup 5^, 5x 104 organisms/ml). The suspensions were made in blood samples with and without anticoagulant and these samples were processed as in the case of clinical specimens for DGM.
Results & Discussion
Ninety two (54.1%) of the 170 patients clinically suspected to have leptospirosis met the standard criteria for diagnosis. Leptospirosis could be isolated in 30 patients and sera from all these patients showed significant titre of antibody.
The results of different tests in the two sets of subjects are summarized in the Table. Among the 92 confirmed cases, DGM detected leptospires in the plasma of 37 patients (40.2%) and in the serum of 31 (33.7%). Among the patients who did not meet the criteria for diagnosis, DGM was positive in the plasma of 30 (38.5%) patients, and in the serum of 31 (39.8%). In contrast the sensitivity and specificity of IgM ELISA and Dipstick were in the range of 80-90 per cent.
The indices of efficacy of DGM were calculated for the results using plasma. The calculated sensitivity of DGM was 40.2 per cent and the specificity was 61.5 per cent. The positive and negative predictive values were 55.2 per cent and 46.6 per cent respectively. The test had 50 per cent agreement with the Gold Standard and the K value of agreement was 0.017141 (Z=0.23, P=0.407705). The two observers had 94.7 per cent agreement and the kappa value of agreement was 0.891382 (Z = 11.62, P
In the 30 patients from whom isolation of leptospira was successful, leptospires could be demonstrated by DGM in 12 giving a positivity rate of 40 per cent among culture positive patients. Eighteen culture positive patients (60%) gave positive results in IgM ELISA and 17 (56.7%) were positive by Lepto-Dipstick during the acute phase. During the convalescent stage the numbers were 26 (86.7%) for ELISA and 25(83.3%) for Dipstick. These IgM based tests have much higher sensitivity than DGM and hence are preferable over DGM for early diagnosis of leptospirosis.
To compare the results of DGM at low and high relative centrifugal force (RCF), the plasma samples that was negative at 1000 g were centrifuged again at 3000 g and examined under DGM. In the confirmed patients sera of four patients that were negative by DGM at 1000 g were found to be positive after centrifugation at 3000 g and among the non lepto spirosis patients, sera of 2 patients which were negative at 1000 g, gave positive results after centrifugation at 3000 g. The differences in sensitivity and specificity at lower and higher RCF were not statistically significant.
A concentration of at least 1x 10^sup 5^ organisms/ml is required for demonstrating leptospires in serum under DGM. At the concentration of Sx 104 organisms/ml, 1-2 leptospires per field could be demonstrated in plasma under DGM. At all concentrations, the number of organisms seen per field was higher in the case of plasma than in the case of serum, indicating that plasma is a better specimen than serum. The specimens were examined at different intervals to estimate the duration up to which leptospires retain motility. The organisms could be identified up to 3 h having typical motility under laboratory conditions. However, this period may vary in real practice due to fluctuating temperature and other environmental factors.
DGM is the method of choice for demonstrating leptospires in cultures but it has limitations as a diagnostic aid. Though the organisms are presumably always present in the blood during the first week of disease, it is rarely present in numbers sufficient to allow detection by direct microscopy. In addition, leptospires are excreted in urine intermittently and the concentration of the organism may be too low. It may give false positive results even in experienced hands due to artifacts like serum proteins and cell fragments being mistaken for leptospires in clinical samples Moreover one may get equivocal results as in majority of the clinical samples, leptospires may not exhibit typical movement, which is one of the important character to differentiate leptospires from spiral artifacts.
In the present study we observed low sensitivity (40.2%), specificity (61.5%) positive predictive value (55.2%) and negative predictive value (46.6%) for DGM. The positivity rate of DGM was found to be very low (40%) among the culture positive samples. The positivity rates of the test were similar among patients who met the standard criteria for diagnosis and those who did not. This indicates that the presence or absence of leptospiral infection does not influence the outcome of the test.
We also observed that the usefulness of differential centrifugation is very limited as the difference in the indices of efficacy of the test at 1000 g and 3000 g were found to be statistically insignificant. In fact, the motility of the organism reduced after centrifugation of the plasma specimens at 3000 g. We observed that reading the results was always subjective as in the majority of plasma samples the number of organism per dark field ranged between 0-2 and there was always doubts about the typical nature of the motility.
Though DGM is quick and an alternate method for early diagnosis of leptospirosis where facilities are not available for performing MAT and ELISA, this technique appears to be unreliable as it showed significant number of false positive and false negative results. It is a known fact that the test has low sensitivity. The present study not only emphasizes this but also show that even the specificity of the test is very low. Therefore the examination of clinical specimen using DGM as the single diagnostic procedure is not recommended in routine practice. New tests like Lepto-Dipstick and Lateral Flow, if available at affordable cost, may reduce the dependency of many laboratories on DGM for diagnosis of leptospirosis.
The authors gratefully acknowledge the help and cooperation of hte doctors and staff of the Primary Health Centre, Manglutan, South Andaman and Sh. Paritosh De. Laboratory Assistant, RMRC, Port Blair for assistance in the laboratory.
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P. Vijayachari, A.P. Sugunan, T. Umapathi & S.C. Sehgal
National Leptospirosis Reference Centre, Regional Medical Research Centre (ICMR), Port Blair, India
Reprint requests: Professor S.C. Sehgal, Director, Regional Medical Research Centre, Post Bag No. 13 Port Blair 744101, India
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