Abstract

Leptospirosis in India is increasingly becoming a health problem, particularly during the monsoons. As kidney is one of the most commonly involved organs in leptospirosis we studied clinical and histopathological features in the kidney in 30 autopsy cases of suspected leptospirosis to determine if there are any changes specific for leptospirosis in the kidney. Subsequently, immunohistochemistry (IHC) for leptospirosis was performed in these cases, of which 20 were positive for leptospiral antigen. Most leptospirosis patients were young males with fever, jaundice, myalgias, haemoptysis and oliguria. Autopsies revealed enlarged kidney and extensive pulmonary haemorrhages in all cases. Kidney sections showed acute tubular necrosis and interstitial nephritis. These changes were not so obvious in non-leptospiral cases. We concluded that in autopsy cases of acute febrile illness; if the kidneys are swollen with acute tubular necrosis and interstitial nephritis coupled with extensive pulmonary haemorrhages, then leptospirosis should be suspected.

Introduction

Leptospirosis is an important zoonotic disease with a wide geographic distribution. Inspite of the first description of the disease in man in the year 1883, an accurate estimate of the disease burden is still lacking worldwide as well as in India.1 More and more cases are being diagnosed in recent monsoons since 2000 and it has become a significant health problem in our country. It accounts for a considerable proportion of cases of pyrexia with features like jaundice, acute renal failure, myocarditis or haemorrhagic pneumonitis. Leptospirosis has been classically described as Weil’s disease however clinical presentations other than of Weil’s disease are more common in India and other parts of the world. As leptospirosis has a broad spectrum of clinical presentations, it causes diagnostic difficulty. There are also difficulties in the laboratory diagnosis of the disease in many parts of this country. There are very few studies in the literature, which focus on histopathological changes in kidney in leptospirosis, inspite of the fact that it is one of the most commonly involved organs.1,2 But again these studies are mostly in the older literature and do not highlight the differences in the histopathological features between leptospiral and clinically similar non leptospiral cases. Most of the renal changes seen in leptospirosis are nonspecific and can be seen in variety of other conditions. Hence we carried out a detaied study of histopathological changes in kidney in confirmed cases of leptospirosis and compared it with cases in which leptospirosis was ruled out by immunohistochemistry.

Aims and Objectives

Our main aims and objectives were to study the clinical profile and histopathological changes in the kidney of autopsy cases of leptospirosis and determine if there are any histopathological changes specific for leptospirosis in the kidneys.

Material and Methods

This is a retrospective study of 30 autopsy cases in a two year period with identical clinical features of fever, jaundice, haemorrhagic diathesis and respiratory distress. In all cases, clinical details were recorded from the case papers. A complete autopsy was performed in all cases and a detailed examination of both gross and microscopy of all organs was carried out. Routine hematoxylin and eosin stain was carried out on the kidney sections cut at 4 microns thickness. All the features present in glomeruli, tubules, interstitium and blood vessels were studied critically. Kidney blocks were sent for Immunohistochemistry (IHC) at Centres for Disease Control and Prevention (CDC), Atlanta. The antibody used for IHC in CDC, was a mixture of 16 reference rabbit polyclonal anti-leptospira antiserum which reacts with most of the pathogenic serovars that cause human leptospirosis. DAKO autostainer and a fast red chromagen reagent were used to perform immunostaining. Further, on availability of the IHC reports, the slides were reviewed to analyze whether histopathologically there are any renal changes specific for leptospirosis. Also, severity of histopathological findings was correlated with certain clinical parameters to assess bad prognostic factors.

Results

All these cases were reported in the monsoon season. History of occupation was available only in 6 cases (1- fisherman, 2- labourer, 1- watchman, 1- painter, 1- carpenter). All of them died after a brief hospital stay. Hence, very limited investigations were performed in most of the cases. Urine examination was not performed in any case. The clinical differential diagnosis in all cases was either, leptospirosis, malaria or hepatitis. 20 of these cases turned out to be Immunohistochemistry (IHC) positive for leptospirosis and 10 were negative. This positivity was noted mostly within the tubules in the kidney sections (Fig. 1). The important clinical, laboratory and autopsy findings in the two set of cases are highlighted in Table 1. It is evident from Table 1 that, leptospirosis cases comprised predominantly of young males and all patients died within one week of presentation (except one). Blood urea nitrogen (BUN) values were available in 10 leptospirosis patients and it ranged from 16 to 128 mg/dl and similarly serum creatinine in 10 leptospirosis cases ranged from 1.1 to 6.8 mg/dl. Amongst the deranged potassium levels, 5 out of 7 cases had hypokalaemia. In our study, meningeal involvement or pancreatitis was not noted in any case of leptospirosis clinically as well as on histopathology. Mild to moderate myocardial inflammation was seen in most of the cases.

Fig. 1 : Thread like and dot like organisms within the renal tubules. (400 x).

Table 1 : Important findings in the leptospirosis IHC positive and negative cases*

*in the bracket are given the actual number of cases and for the laboratory investigations, as they were not available in all cases, the percentage is given from the number of cases in which they were performed.

The cause of death in 10 negative cases, were mostly related to the liver and bleeding diathesis.

Grossly, though the actual kidney size was not recorded in any case, considering the weight it is evident that the kidneys were markedly swollen (Fig. 2). Capsule was stripped easily and subcapsular surface was smooth but showed marked capillary congestion. On cut section, the corticomedullary demarcation and ratio was well maintained and medulla was congested.

Fig. 2 : Markedly swollen kidneys in leptospirosis with marked capillary congestion.

Prominent cortical striations were noted. Pelvicalyceal system was unremarkable. 1 out of 20 leptospirosis cases showed subcapsular and pelvicalyceal system haemorrhages in the kidneys, whereas 4 out of 10 non-leptospiral cases showed haemorrhages in the pelvicalyceal system of the kidney.

Microscopy

All 20 leptospirosis cases showed variable changes in the glomeruli within the same case. There was varying degree of glomerular capillary tuft dilatation with congestion and mild to moderate increase in mesangial matrix and cellularity in 19 cases. In 10 cases, there were polymorphs within the mesangium or adherent to the capillary walls within the glomeruli. In 12 cases there was definite periglomerular mixed inflammatory infiltrate noted (Fig. 3). None of the cases showed crescents or any thrombi in the glomeruli. Significant tubular dilatation was noted in 19 cases (Fig. 4). Tubules in 14 cases showed patchy to severe acute tubular necrosis in proximal as well as distal tubules. 7 cases showed loose collections of polymorphs and nuclear debris in the tubules and few cases showed granular, hyaline and pigment casts (Fig. 5). There was varying degrees of interstitial oedema in 16 cases and

Fig. 3 : Periglomerular mixed inflammatory infiltrate (400 x).	Fig. 4 : Tubular dilatation and interstitial oedema (100 x).	Fig. 5 : Loose collections of polymorphs within the tubules (400 x).

all cases showed diffuse inflammatory infiltrate of lymphocytes, plasma cells, macrophages, polymorphs and occasionally eosinophills in the interstitium. Inflammation was more prominent in cortex than in the medulla. Only one case showed focal interstitial haemorrhage. There was extreme congestion of the peritubular capillary network with occasional margination of polymorphs within them. Blood vessels, apart from congestion, did not show vasculitis or any other change. 10 cases showed dilatation of lymphatic channels.

When 6 oliguric cases were compared to 14 non-oliguric cases, acute tubular necrosis and casts were seen in all oliguria cases compared to 8 out of 14 cases in the non oliguric patients. When the severity of histopathological changes in the kidneys were compared to blood urea nitrogen or creatinine levels and platelet counts, no correlation was found.

When compared to the 10 non-leptospiral cases, certain subtle changes were noted which are depicted in Table 2. Thus, Table 2 depicts that certain features like mesangial hypercellularity, periglomerular inflam-matory infiltrate, tubular dilatation, interstitial oedema and nephritis are commonly observed in cases of leptospirosis as compared to other causes of acute febrile illness with loose collections of polymorphs within the tubules seen only in cases of leptospirosis.

Discussion

Leptospirosis is one of the important cause of acute renal failure (ARF) in a setting of pyrexia in India.3 We have compared our

Table 2 : Comparison of microscopic findings in the leptospiral and non-leptospiral cases*

* in the bracket are given the actual number of cases.

results with those noted in other cities of South East Asia.3-6 Leptospirosis is commonly seen in young males after heavy monsoon rains leading to water logging and flooding as in our study.1,3-6 Common clinical presentations are fever, jaundice, oliguria, myalgias, conjunctival suffusions and haemorrhagic tendencies.1,3-6 In our autopsy study, fever, myalgias and jaundice were one of the most common clinical presentations. In certain studies, oliguria is also a common complaint in leptospirosis, which is not so in our study, where oliguria was reported in 6 out of 20 cases.1,5 There are two phases of leptospiral infection, initial ‘septicaemic’ phase and subsequent ‘immune’ phase.3,7 In fatal cases there might not be progression to the second phase, which explains absence of meningitis in our study which is thought to be due to immune mechanisms.7

Due to short duration of hospital stay, adequate laboratory investigations were not available in any of the case and this is one of the drawbacks of our study. Mild leucocytosis, thrombocytopenia, mildly elevated liver enzymes, azotaemia and electrolyte derangements seen in our cases, have been reported in other studies also.1,5-7 In one study, days of hospitalization correlated with peak creatinine levels and platelet nadir but we could not demonstrate any such correlation.6 Hence we could not comment on any bad prognostic factors.

Kidney is one of the most commonly involved organs in leptospirosis. Grossly, the kidneys have been reported to be markedly swollen due to interstitial oedema, as also noted in our cases.1,3,5,6 However cortical widening and haemorrhages reported in the kidneys by Arean, was not seen in our study.1 Microscopy revealed that certain features like mesangial hypercellularity, acute tubular necrosis, interstitial oedema and nephritis with a definite periglomerular location are commonly noted in cases of leptospirosis. Though these changes have been reported in scattered studies of literature, the exact incidence of these changes in leptospirosis has not been reported.1,2 The margination of polymorphs in capillaries in kidney indicates that our cases were in the initial septicaemic phase of the disease. In literature, vasculitis has also been reported, but was not seen in our cases.8 These changes are reversible if the infection is controlled.5

Coming to the mechanisms involved in leptospirosis which lead to these renal changes, we can say that azotaemia is due to pre-renal causes like decreased renal perfusion as 15 out of 20 cases presented in a shock like state with tachycardia and hypotension.7 Acute tubular necrosis (ATN) is due to ischaemia as well as due to endotoxins released by leptospiral organisms.3,5 Organisms initially cause glomerular injury through blood circulating in it and then via peritubular capillary network, it reaches the interstitium causing interstitial oedema and nephritis and finally cause tubular damage.3,5 Tubular epithelium, when exposed to outer membrane proteins of leptospiral organisms, activate nuclear NFkB binding and stimulate nitric oxide, monocyte chemoattractant protein-1, tumour necrosis factor-a which also play an important role in developing tubulo-interstitial nephritis.5 Hence in rapid fulminant cases, acute interstitial nephritis may be absent all together. The varying degree of interstitial nephritis seen in our cases could be due to this reason. Acute interstitial nephritis and acute tubular necrosis are causes of acute renal failure, which is seen in all our cases.5

Over all mortality rate in leptospirosis is reported around 20.8%, however our study cannot provide any such data as we did not have any statistics about the actual incidence of all clinical cases of leptospirosis.3 In our study, extensive pulmonary haemorrhage was the major cause of death in our cases as reported in some other studies.3,4,9 This is due to capillary fragility, which is a characteristic feature of this disease and it is presumed to be secondary to immune mediated vasculitis probably due to antineutrophil cytoplasmic autoantibodies (ANCA).4 But this requires to be further studied. Also, the infection or any other disease which caused death in the 10 non leptospiral cases need to be confirmed.

To best of our knowledge, immuno-histochemistry is not used in studies we came across to make the diagnosis of leptospirosis, which we have used as we did not have the backup of routine serological tests. This technique needs to be further explored to be useful routinely and exact specificity and sensitivity rates should be determined. This is one drawback of this study, though we have relied on it to make the diagnosis of leptospirosis. Further studies are also required to find out the type of immunological response which comes into play in the initial phase of the disease to prevent deaths due to leptospirosis.

Hence, to summarize, kidney is one of the most commonly involved organ in leptospirosis, however all changes seen in the kidney are non-specific. But, in autopsy cases of acute febrile illness with no serological backup, if features like swollen kidneys, mesangial hypercellularity, acute tubular necrosis and acute interstitial nephritis with definite periglomerular location are noted, especially in monsoons, leptospirosis should be suspected in presence of extensive pulmonary haemorrhages.

Acknowledgements

We are thankful to Centres of Disease Control and Prevention, Atlanta for having helped us out with the Immunohistochemistry of our cases.

Ethical clearance: As these are autopsy cases, relative permission and necessary clearance was already taken during the autopsies.

References

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7. Peter Speelman. Chapter 174. In Harrison’s principles of Internal Medicine. 15th edition. MacGraw Hill Publications; 2001. pp 1055-57.
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9. Zaki SR, Sheih W-J. Leptospirosis associated with outbreak of acute febrile illness and pulmonary hemorrhage, Nicaragua, 1995. Lancet 1996; 347 : 535-36.

*Lecturer; **Associate Professor; ***Professor and Head, Department of Pathology, T N Medical College and B Y L Nair Ch . Hospital, Mumbai Central, 400008.