Orientia, rickettsia, and leptospira pathogens as causes of CNS infections in Laos: a prospective study

Summary

Introduction

The most common bacterial pathogens responsible for meningitis in southeast Asia are Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, S suis, and Mycobacterium tuberculosis.¹ Timely empirical and specific pathogen-directed treatment is essential, usually, except for M tuberculosis, including a third-generation cephalosporin. However, many patients with CNS infections do not receive a causal diagnosis despite cerebrospinal fluid (CSF) culture and DNA molecular assays,² partly because of low CSF pathogen density and previous antibiotic use.² Other neglected bacteria probably cause CNS infections in Asia, including pathogens not expected to respond to third-generation cephalosporins. During World War 2, scrub typhus (caused by Orientia tsutsugamushi) was a well-recognised cause of lethal meningitis in the Asia-Pacific region, but this clinical experience has largely been forgotten.³ Findings of studies in India and Thailand showed that up to 15% of patients with scrub typhus had neurological complications.^{4, 5, 6} O tsutsugamushi DNA was detected in the CSF of Taiwanese patients serologically confirmed to have scrub typhus.⁷ Indeed, altered CNS function is implicit in the name typhus, which means stupor. Similarly, Rickettsia typhi (the cause of murine typhus) and other Rickettsia species cause meningoencephalitis. Another neglected but common group of pathogens, the Leptospira spp have received little attention as causes of CNS infection. In a study in the Philippines, 5% of patients with aseptic meningitis had high serological titres to Leptospira spp;⁸ in a Brazilian study, more than 50% of patients with aseptic meningitis were CSF PCR positive for Leptospira spp.⁹ However, leptospiral meningitis would be expected to respond to third-generation cephalosporins, if severe leptospirosis does respond to antibiotics.¹⁰

Leptospira and Rickettsia species are distributed worldwide^{11, 12} and O tsutsugamushi is endemic across Asia, the Pacific islands, and northern Australia.¹³ Although a vast human population is potentially exposed to treatable rickettsial and leptospiral diseases, few data exist on the incidence and clinical features of rickettsial and leptospiral CNS infections. Appropriate diagnostic methods or trialled optimum treatments are scarce. Scrub typhus, leptospirosis, and murine typhus are common diseases in Laos, both in the capital, Vientiane, and in rural areas^{14, 15} and in adjacent countries, including China and Thailand. Although Asia is geographically, culturally, economically, and ethnically diverse, Laos is an example of the vast areas of rural Asia that are economically poor with little medical information to guide public health policy. Therefore, we assessed whether these pathogens are important causes of CNS infections in Laos.

Methods

Results

Between 2003 and 2011, 1112 patients were recruited and physicians collected CSF from 1051 (95%) patients (appendix p 1). Most patients were male and had a history of antibiotic administration (table 1). Of the 1051 patients with CSF samples available, 254 (24%) had evidence of bacterial (194 [76%]) or fungal (60 [24%]) infection, of which 90 (35%) were attributed to O tsutsugamushi, R typhi/Rickettsia spp, or Leptospira spp (figure 1). By PCR, serology, and culture, 45% of inpatients who had a lumbar puncture were assigned a bacterial, fungal, or viral laboratory diagnosis (Dubot-Pérès A, et al, unpublished).

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Figure 1

Summary of diagnosed bacterial and fungal infections

Conventional bacteria were defined as Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, or S suis.

42 (4%) of 1051 patients had culture or PCR evidence of a monoinfection with a conventional bacterial pathogen: S pneumoniae (25 [2·4%]), N meningitidis (4 [0·4%]), H influenzae (9 [0·9%]), and S suis (4 [0·4%]). By comparison, 90 (8·6%) of 1051 patients had culture, PCR, or serological evidence for monoinfections with O tsutsugamushi, R typhi/Rickettsia spp, or Leptospira spp (table 1). The frequency of these combined pathogens (8·6%) was significantly higher than that for conventional bacteria (4·2%; p<0·0001).

Patients with conventional bacterial meningitis were a similar age to those with scrub typhus, but patients with R typhi or Rickettsia spp and Leptospira spp infections tended to be older (table 1). Most (879/1112 [81%]), patients were residents of Vientiane City and Vientiane Province, reflecting the hospital catchment area and was similar for all study groups. No district or occupation was associated with higher patient numbers.

With PCR or culture assays, detection of moninfections with O tsutsugamushi, R typhi or Rickettsia spp, or Leptospira spp infection ranged from 1·9% to 2·9% (table 2). Of 446 patients with data available for both direct and serological results, we detected evidence of scrub typhus, Rickettsia spp or murine typhus, or leptospirosis in 59 (13%) of 446 patients. Leptospira interrogans was the most commonly identified Leptospira species by microscopic agglutination test or sequencing (GenBank KJ150298-KJ150302; appendix p 3). We identified Rickettsia spp as R typhi for 16 (84%) of 19 patients who were qPCR positive, but for three patients the Rickettsia species could not be established because no template remained. In addition to the 90 patients with O tsutsugamushi, R typhi/Rickettsia spp and Leptospira spp monoinfections, we recorded grade 1 and grade 2 multiple infections in an additional 14 patients (appendix p 4).²⁴

More than half of all patients met the WHO criteria for meningitis (709/1093 [65%]) or acute encephalitis syndrome (610 [56%]), 84 (74%) met either criteria, and 521 (48%) fulfilled both criteria (table 1 and figure 2). Patients with a conventional bacterial CNS infection presented with the shortest median duration of fever (p=0·005), the highest frequencies of convulsions (p=0·023) and neck stiffness (p=0·182), the lowest median Glasgow coma scale (GCS) scores on admission (p=0·062), and the highest mortality (p=0·076), compared with patients with R typhi or Rickettsia spp and Leptospira spp infections (table 1).

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Figure 2

Patients who fulfilled WHO criteria¹⁷ for meningitis, meningoencephalitis, AES, or none of these criteria

Meningoencephalitis is defined as fulfilling criteria for both meningitis and AES. AES=acute encephalitis syndrome.

Patients with a CNS infection caused by R typhi/Rickettsia spp tended to present later in their illness than patients with conventional bacteria (table 1). They had the lowest frequency of vomiting and none reported photophobia. The mortality of patients with R typhi or Rickettsia spp (27%) was nearly double that of patients with scrub typhus (14%, p=0·307) and leptospirosis (13%, p=0·299); however, these differences were not significant. Notably, the mortality of patients with R typhi/Rickettsia spp was similar to those infected with conventional bacteria (about 33%). Furthermore, compared with patients with conventional bacteria, a similar proportion of patients presented with a reduced GCS score (table 1).

Patients with O tsutsugamushi also presented late in their illness and had a high frequency of rash (20%; table 1). 81% fulfilled the WHO meningitis criteria (figure 2) with a significantly higher median GCS score and the highest reported preadmission antibiotic use (86%) of investigated groups.

Fewer patients in the Leptospira group had convulsions (19%) than those in the conventional bacterial group (44%) and all other investigated groups, but presented with the highest frequency of peripheral neurological abnormalities (11%). One patient had GCS 15/15 and bilateral limb weakness (Medical Research Council [MRC] power 2/5) without knee or ankle reflexes and unrecorded sensation; one had GCS 15/15 and bilateral limb weakness (MRC power 4/5) with unrecorded reflexes and sensation; and a third had GCS 9/15, convulsions, and right leg weakness (MRC power 4/5) with normal reflexes and unknown sensation.

Patients with O tsutsugamushi or R typhi or Rickettsia spp infections presented with significantly longer fever duration (median 6 days, range 1–30 vs 4 days, 0–120; p=0·004), and significantly more commonly had a rash (9/58 with O tsutsugamushi or Rickettsia spp and 3/73 with Leptospira spp or conventional bacteria infection; p=0·033) compared with patients with conventional bacteria and leptospira. In multivariate analysis, we identified no independent clinical risk factors for O tsutsugamushi or R typhi/Rickettsia spp infections. The mortality in this combined group (10/48 [21%]) was not significantly different from patients with conventional bacterial infection (p=0·189).

Patients with conventional bacterial infections had high opening pressures, frequent CSF turbidity, and high cellularity. Cells were mainly neutrophils, with high CSF lactate, low glucose, and high protein concentrations (table 1).

Despite similar clinical severity and mortality, the CSF characteristics of patients infected with R typhi or Rickettsia spp differed from the conventional bacteria group. The opening pressure tended to be lower and only one patient had CSF turbidity. CSF cellularity, neutrophil to lymphocyte ratio, and lactate and protein concentrations were also significantly different from conventional bacterial infections (table 1).

For patients infected with O tsutsugamushi, opening pressures were similar to those with conventional bacterial illness, but CSF turbidity and cellularity were significantly less common and CSF lactate concentrations were the lowest of all groups (table 1). Compared with those with R typhi or Rickettsia spp infections, patients with scrub typhus had significantly higher CSF white cell counts (p=0·018) and a two-fold higher neutrophil to lymphocyte ratio (p=0·045).

For patients infected with Leptospira spp, the CSF opening pressure was similar to the other groups, but CSF turbidity and cellularity were rare, with lower protein and higher glucose concentrations than patients in the conventional bacteria group (table 1).

Visual turbidity (5/50; p=0·002) and cellularity (median 38·5 cells per mm³, range 0–653; p=0·0001) were significantly less frequent for patients with O tsutsugamushi or R typhi/Rickettsia spp infections, than for patients with Leptospira spp or conventional bacterial infections (turbidity 24/70; median cellularity 235 cells per mm³, range 0–9, 600). Compared with O tsutsugamushi or R typhi/Rickettsia spp infections (median neutrophil to lymphocyte ratio 1·0, range 0–19), patients with conventional bacteria and Leptospira spp infections had two times higher neutrophil to lymphocyte ratios (median ratio 2, range 0–174; p=0·011). Eight (19%) of 42 patients with O tsutsugamushi or R typhi/Rickettsia spp infections presented with CSF glucose lower than 2·5 mmol/L compared with 30 of 51 patients in the combined Leptospira spp and conventional bacterial infection group. 12 (29%) of 42 patients with O tsutsugamushi or R typhi/Rickettsia spp infections had lactate concentrations higher than 4 mmol/L compared with 39 (67%) of 58 patients in the combined Leptospira spp and conventional bacterial infection group (p<0·0001). A lower white cell count was associated with an increased odds of O tsutsugamushi or R typhi/Rickettsia spp infection; the odds decreased 1% with every unit increase in white cell count (OR 0·997, 95% CI 0·995–0·999).

Infections with conventional bacteria showed a distinct seasonal pattern, peaking in the dry (November–April) season (dry: 26/441 [6%], wet: 15/655 [2%], p=0·002; figure 3). By contrast, O tsutsugamushi was diagnosed with greater frequency in the wet season (27/655 [4%]) than the dry season (9/441 [2%], p=0·055). The frequency of R typhi/Rickettsia spp and Leptospira spp did not significantly differ between seasons (R typhi/Rickettsia spp; dry: 17/655 [3%]; wet: 16/441 [4%], p=0·369; Leptospira spp; dry: 26/655 [2%]; wet: 13/441 [3%], p=0·371).

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Figure 3

Monthly proportions of CNS patients diagnosed with Orientia tsutsugamushi, Rickettsia typhi/Rickettsia spp, Leptospira spp, or so-called conventional bacterial infections among all patients recruited with suspected CNS disease, 2003–11

Total number of patients recruited per month during this study, 2003–11, is shown by bars corresponding to the indicated pathogens. The line represents the mean rainfall (mm) per month in Vientiane during the investigated years. Data from Department of Meteorology and Hydrology, Ministry of Natural Resources and Environment, Lao PDR. *Patient recruitment to this study stopped in November, 2011.

17 (55%) of 31 patients with O tsutsugamushi were treated with appropriate antibiotics compared with 11 (39%) of 28 with R typhi/Rickettsia spp infections (table 3). Ten (17%) of 59 patients with O tsutsugamushi or R typhi/Rickettsia spp infection died, of whom three received appropriate treatment. In this small sample, we noted no significant association between patients receiving appropriate treatment and outcome (p=0·179). Nearly all (30/31) patients with a Leptospira spp infection received appropriate treatment with doxycycline, chloramphenicol, a cephalosporin, or penicillin alone (14/30) or in combination (16/30). All three patients of the 24 patients with fatal leptospirosis received appropriate treatment.

Discussion

These data suggest that O tsutsugamushi, R typhi or Rickettsia spp, and Leptospira spp infections are the leading causes of bacterial CNS infections in Laos. With increasing evidence that O tsutsugamushi, R typhi or Rickettsia spp, and Leptospira spp are important causes of fevers, these findings raise concerns that these infections are responsible for a large proportion of neglected but treatable CNS disease burden in the many endemic countries and in travellers (panel).^{12, 14, 25, 26}

8% of all CNS infections and 46% of identified bacterial causes were attributable to O tsutsugamushi, R typhi/Rickettsia spp, or Leptospira spp, with conservative and robust diagnostic definitions; more than double the 22% attributed to four conventional bacteria species. The overall mortality of CNS infections in Laos was 27%, with group-specific mortality of 18% for Orientia, Rickettsia, and Leptospira spp, and 33% for conventional bacteria, which emphasises the importance of improving diagnostic and treatment strategies. Comparisons of clinical findings highlight differences that might serve as diagnostic clues. For example, patients with R typhi/Rickettsia spp and Leptospira spp infections were older and patients with O tsutsugamushi presented later in their illness, consistent with reports from India,⁶ and commonly had skin rashes. Elsewhere, hearing loss has often been noted in patients with O tsutsugamushi,²⁷ but was not significantly more common in patients with O tsutsugamushi and CNS disease in Laos than other patient groups. Consistent with previous findings,⁶ patients with O tsutsugamushi and R typhi/Rickettsia spp infections presented with low, but abnormal CSF white cell counts; turbid CSF and raised CSF lactate concentrations were infrequent.

Data from recent reports suggest that admission interstitial pneumonitis is associated with meningitis in patients with O tsutsugamushi infection.^{6, 28} Although most Lao patients with O tsutsugamushi presented with tachypnoea, this was not significantly more frequent than in other groups (p=0·587). Furthermore, O tsutsugamushi infection commonly presents with tachypnoea without CNS involvement.¹⁵ R typhi/Rickettsia spp infections were more severe than O tsutsugamushi and leptospirosis in terms of GCS and mortality. However, R typhi infection is generally regarded as benign²⁹ and although it is distributed worldwide, is rarely included in the differential diagnosis of CNS disease.

Reduced consciousness and seizures were the most common neurological symptoms in Indian patients with neuroleptospirosis.³⁰ We did not record this finding in Laos, which raises questions about variation in strain virulence and host susceptibility. Three patients with leptospirosis had some neurological evidence of intracerebral lesions, consistent with the putative association between leptospirosis and Moyamoya disease, but cerebral angiography, which is not available in Laos, is needed to confirm the diagnosis.³¹ Notably, only patients with leptospirosis had abnormal peripheral neurological symptoms and signs.

Conventional bacterial infections were significantly associated with turbid, cellular CSF containing high neutrophil counts. The high frequency of CSF abnormalities (including increased white cell counts, turbidity, and lactate concentrations) in all patients who had a lumbar puncture, but without a causal diagnosis, suggests that many of these patients had undetected infections and that patients with important CNS pathology are not receiving lumbar punctures.

During this 9 year investigation, we noted a distinct seasonal pattern, similar to findings for non-malarial fevers in Laos.¹⁴ Conventional bacterial infections (eg, N meningitidis and S pneumoniae), were most frequent in the dry season, peaking in January, consistent with data from India,³² whereas Orientia, Rickettsia, and Leptospira species were detected in nearly 20% of febrile patients diagnosed with CNS disease at the end of the rainy season. This seasonality and CSF characteristics could help to guide clinicians' differential diagnosis.

Our study has important limitations, including the use of suboptimum samples for leptospiral culture,³³ the known limitations of rickettsial diagnostics,^{34, 35} and the use of stored samples. However, these limitations probably led to the underestimation of the incidence of Leptospira spp, O tsutsugamushi, and R typhi/Rickettsia spp infections. The widespread use of over-the-counter antibiotics² before admittance to hospital probably reduced culture rates for conventional bacteria, and we did not do serology assays for these pathogens. The lack of clustered results and stringent sample handling protocols, physical separation of processes, and the use of uracil-DNA glycosylase in the PCR mix, makes specimen contamination very unlikely. The three Rickettsia spp infections that could not be speciated were probably R typhi because this is the main species in Laos.^{14, 36}

Our data suggest that empirical treatment practice for CNS infections in Laos, where third-generation cephalosporin monotherapy is generally used, should be reconsidered. Although current guidelines are probably effective for leptospiral CNS disease, there is no evidence for their efficacy against O tsutsugamushi and R typhi/Rickettsia spp, which should be treated with doxycycline or chloramphenicol. Azithromycin or rifampicin might be active against O tsutsugamushi CNS disease.³⁷ More data are needed for CSF drug levels in patients with typhus and variability in minimum inhibitory concentrations against R typhi/Rickettsia spp and O tsutsugamushi, with clinical trials to inform optimum treatment.²⁸ Because of the paucity of accessible and accurate admission laboratory typhus diagnostics,^{34, 38} empirical treatment with doxycycline plus a third-generation cephalosporin might be appropriate in areas endemic for scrub typhus and murine typhus. However, combination of bacteriostatic tetracyclines with bactericidal cephalosporins might reduce the efficacy in typhus-endemic areas for treatment of conventional bacterial pathogens, such as S pneumoniae.³⁹ The optimum management of patients with either confirmed O tsutsugamushi, R typhi/Rickettsia spp, or Leptospira spp CNS infection or of those in which these or conventional bacteria are suspected remains unclear, with little evidence to guide policy. Indeed, there is very little evidence on the pharmacokinetics of the tetracyclines in CSF, or optimum dose for CNS disease.⁴⁰ These findings suggest that greater appreciation and further investigation of Orientia, Rickettsia, and Leptospira spp as neglected but treatable causes of CNS disease in other endemic areas globally is urgently needed.^{3, 41} Although pathogen discovery has an important role, we suggest that optimising diagnosis, treatment, and prevention of these neglected but common bacteria might have a more rapid beneficial public health outcome.

Acknowledgments

Contributors

Declaration of interests

References