Serological Analysis by Enzyme-Linked Immunosorbent Assay Using Recombinant Antigen LipL32 for the Diagnosis of Swine Leptospirosis

Leptospirosis is an important global zoonotic disease caused by pathogenic Leptospira spp. species. Swine leptospirosis has a major economic impact because pigs are sources of animal protein and by-products. The signs of swine leptospirosis are abortion, stillbirth, birth of weak or ill piglets, appearing 14–60 days after infection. The reference method for diagnosis of leptospirosis is the microscopic agglutination test (MAT), in which serum samples are reacted with live antigen suspensions of leptospiral serovars. However, MAT is laborious and time consuming as a diagnostic procedure when dealing with a large number of samples; therefore, efforts are being made to develop novel, sensitive, and specific diagnostic tests for leptospirosis. In this study, a recombinant LipL32 based on enzyme-linked immunosorbent assay (rLipL32/ELISA) was evaluated as a screening test for the detection of pathogenic leptospiral-specific antibodies. A total of 86 swine serum samples tested by MAT were used to develop rLipL32/ELISA. Compared to positive and negative sera tested by MAT, rLipL32/ELISA showed 100 % sensitivity, 85.1 % specificity, and 91.86 % accuracy. No positive reaction for other bacterial diseases (enzootic pneumonia and brucellosis) was observed. The rLipL32/ELISA reported in this study is a specific, sensitive, and convenient test for the detection of antibodies against swine leptospiral infection and can be used as a rapid screening test in epidemiological surveys.     

Comparison of conventional PCR, quantitative PCR, bacteriological culture and the Warthin Starry technique to detect Leptospira spp. in kidney and liver samples from naturally infected sheep from Brazil

Leptospirosis is an infectious disease of worldwide importance. The development of diagnostic techniques allows sick animals to be identified, reservoirs to be eliminated and the disease prevented and controlled. The present study aimed to compare different techniques for diagnosing leptospirosis in sheep. Samples of kidney, liver and blood were collected from 465 animals that originated from a slaughterhouse. The sera were analyzed by the Microscopic Agglutination Test (MAT), and kidney and liver samples of seropositive animals were analyzed using four techniques: bacteriological culture, the Warthin Starry (WS) technique, conventional PCR (cPCR), and quantitative PCR (qPCR). With the MAT, 21 animals were positive (4.5%) to serovars Hardjo (n=12), Hebdomadis (n=5), Sentot (n=2), Wolfii (n=1) and Shermani (n=1). Titers were 100 (n=10), 200 (n=2), 400 (n=6) and 1600 (n=3). No animal was positive by bacteriological culture; four animals were positive by the WS technique in kidney samples; six animals were positive by cPCR in kidney samples; and 11 animals were positive by qPCR, eight of which in kidney samples and three in liver. The bacterial quantification revealed a median of 4.3 bacteria/μL in liver samples and 36.6 bacteria/μL in kidney samples. qPCR presented the highest sensitivity among the techniques, followed by cPCR, the WS technique and bacteriological culture. These results indicate that sheep can carry leptospires of the Sejroe serogroup, and demonstrate the efficiency of quantitative PCR to detect Leptospira spp. in tissue samples.     

Recombinant ligA for leptospirosis diagnosis and ligA among the Leptospira spp. clinical isolates

Rapid diagnosis for differentiation of leptospirosis from other pyrogenic infections prevailing in the same locality is imperative for proper treatment. During infection, the pathogenic Leptospira spp. express virulence factors which induce antibody responses in the infected host. In this study, 50 referenced Leptospira spp. belonging to six genomospecies and 10 L. interrogans clinical isolates were studied for the presence of a gene encoding an in vivo expressed, surface exposed, immunoglobulin-like protein, LigA, by using PCR and southern hybridization specific to the 5' terminus sequence of the DNA. LigA was also detected in the Leptospira spp. whole cell homogenates by a direct ELISA using a mouse antiserum to the C-terminal portion of recombinant LigA (cLigA) as a detection reagent. All pathogenic Leptospira spp. except one of the two strains of L. santorasai were positive for the gene and its phenotype while all of the L. borgpetersenii and L. biflexa strains were negative. Recombinant cLigA was used as an antigen in ELISAs for detecting IgM and IgG in the sera of leptospirosis patients and in the sera of patients with other febrile illnesses and healthy subjects. When acute phase sera were tested by the cLigA IgM- and IgG-ELISAs, 92% and 100% of the MAT-positive sera were positive, respectively. The diagnostic sensitivity was 100% when both IgM- and IgG-ELISAs were performed on the same acute phase sera and the results were combined. Acute and convalescence sera of patients who were Leptospira culture positive but MAT/IgM-dipstick negative gave 88% and 100% positives by combined cLigA IgM/IgG ELISAs. The diagnostic specificities for the cLigA IgM- and IgG-ELISAs were 98% and 100%, respectively. Our cLigA based-serology has a high potential for early diagnosis of leptospirosis especially when the culture and MAT results are not yet available.     

Diagnostic method-based underestimation of leptospirosis in clinical and research settings; an experience from a large prospective study in a high endemic setting

BackgroundLeptospirosis has globally significant human mortality and morbidity, yet estimating the clinical and public health burden of leptospirosis is challenging because timely diagnosis remains limited. The goal of the present study was to evaluate leptospirosis undercounting by current standard methods in both clinical and epidemiological study settings.Methodology/Principal findingsA prospective hospital-based study was conducted in multiple hospitals in Sri Lanka from 2016 to 2019. Culture, whole blood, and urine samples were collected from clinically suspected leptospirosis cases and patients with undifferentiated fever. Analysis of biological samples from 1,734 subjects confirmed 591 (34.1%) cases as leptospirosis and 297 (17.1%) were classified as “probable” leptospirosis cases. Whole blood quantitative PCR (qPCR) did identify the most cases (322/540(60%)) but missed 40%. Cases missed by each method include; urine qPCR, 70% (153/220); acute sample microscopic agglutination test (MAT), 80% (409/510); paired serum sample MAT, 58% (98/170); and surveillance clinical case definition, 53% (265/496). qPCR of negative culture samples after six months of observation was of diagnostic value retrospectively with but missed 58% of positives (109/353).ConclusionLeptospirosis disease burden estimates should consider the limitations of standard diagnostic tests. qPCR of multiple sample types should be used as a leading standard test for diagnosing acute leptospirosis.     

Nested polymerase chain reaction for detection of pathogenic leptospires

Leptospirosis is a widespread zoonosis caused by pathogenic members of the genus Leptospira that has a great impact on human and veterinary public health. Early diagnosis of leptospirosis is important because severe lepto spiral infection can have a fulminant course. The available serological techniques for the diagnosis of leptospirosis have low sensitivity during the early stage of the disease. Efforts are being made to develop simpler, effective, efficient, and inexpensive diagnostic methods. In this work, we first evaluate a polymerase chain reaction (PCR) based method for diagnosis of leptospirosis. Primers were designed to amplify a 264 bp region within the lipL32 gene that is conserved among pathogenic Leptospira and absent in nonpathogenic species. The sensitivity and specificity of the assay were evaluated using 7 saprophytic serovars, 37 pathogenic serovars, and 15 other microorganisms. The method was very specific for pathogenic serovars, however, it lacked sensitivity. To enhance the sensitivity, another primer pair was designed to amplify a 183 bp region within the 264 bp region of the lipL32 gene and was used in a nested PCR assay. This approach was much more sensitive than conventional PCR.     

Development and evaluation of latex agglutination test coating with recombinant antigen,LipL32 for serodiagnosis of human leptospirosis

Leptospirosis is a widespread zoonotic disease caused by Leptospira interrogans. Symptoms of disease range from mild symptoms to serious complications including, jaundice, pulmonary hemorrhage, renal and hepatic failure, which may prove fatal. Clinical presentations of this disease are similar with other febrile illness. Therefore, rapid and appropriated laboratory diagnostic tests are needed to aid clinical case identification. As these reasons, objective of this study is to develop and evaluate a simple latex agglutination test coating with recombinant leptospiral antigens, LipL32 for serodiagnosis of human leptospirosis. Firstly, lipl32 gene was amplified from genomic DNA of Leptospira interogans serovar Pyrogenes. Then PCR product of lipl32 gene was ligated with pGEX-2T plasmid, generating pGRK32 recombinant plasmid. Recombinant GST-LipL32 protein was overexpressed and subsequently purified by using Glutathione-Agarose Resin. Recombinant GST-Lipl32 protein was coated on latex beads for development latex agglutination test (LAT). The relative sensitivity, specificity and accuracy of the developed LAT were compared with indirect immunofluorescences assay (IFA) for detection of anti-leptospiral antibodies in 30 human leptospirosis samples, 30 healthy blood donor samples, 10 dengue fever positive samples, 10 scrub typhus positive samples, and 10 melioidosis samples. Results showed that the developed LAT showed sensitivity, specificity and accuracy: 66.66%, 86.66%, and 80.00%, respectively, comparing with IFA method. Moreover, Kappa analysis showed agreement rate of the two methods were 0.421. It concluded that our developed gave compatible result with IFA. Additionally, Our LAT are simple, rapid and suitable for detection in the field. However, for better sensitivity, diagnostic specificity, positive predictive value, negative predictive value, accuracy and Cohen’s kappa comparison should be done in larger amounts of sera samples.     

Evaluation of an IgM-ELISA test for the diagnosis of human leptospirosis

Leptospirosis is a zoonosis with a worldwide distribution very common in most countries. In Italy this acute febrile illness is more frequent in the Northern than in the Southern regions. In the period 1994-1996, the number of cases of Leptospirosis in Sicily was lower with respect to the northern-central regions (7.2% and 73.4% respectively). Between January 1990 and December 1999, a total of 9 leptospirosis cases were observed in the Regional Centre for Leptospirosis of Palermo. The patients were all males (age between 22 and 59 years) and their occupations varied. Laboratory diagnosis is performed by the classical microagglutination microscopical (MAT) but this test is very complex and time-consuming. This study compared the classical MAT with ELISA IgM by using 19 serum samples from 9 patients with confirmed leptospirosis. We also tested 23 serum samples from blood-donors and 29 serum samples from patients with other infectious diseases. By the MAT and the PanBio IgM ELISA all sera from patients were found to be positive. Our results indicate that MAT represents the test with the highest degree of specificity (100%), but ELISA is simpler to perform, considering the favourable degree of sensitivity (100%) and specificity (95.9%).     

Evaluation of MAT, IgM ELISA and PCR methods for the diagnosis of human leptospirosis

Serum samples from 125 patients with clinical suspicion of leptospirosis were tested by the microscopic agglutination test (MAT), IgM ELISA and PCR for the diagnosis of the disease. Most patients were adult males and 74.1% (p<0.001) of the patients exposed to water and 77.6% (p<0.001) of those exposed to animals, were respectively considered confirmed or probable cases by MAT. The clinical symptoms mainly observed among the patients considered confirmed or probable cases were fever (95.6%), jaundice and headache (79.4%), myalgia (77.9%), nausea and vomiting (64.7%). About 63% of the confirmed or probable cases were patients that lived in Belo Horizonte, a big city of the Minas Gerais state, Brazil, showing the occurrence of urban leptospirosis. Among the 47 confirmed cases of leptospirosis diagnosed by MAT, 44 (94%) serum samples were positive by IgM ELISA and 17 (36%) were PCR positive. Among the 33 probable cases, 10 (30%) samples showed positive amplification by PCR. By considering MAT as the standard test, the sensitivity and specificity of IgM ELISA was 96.6% and 93.3%, respectively. A relevant finding in our study was the number of positive cases verified by PCR (13-29%) and IgM ELISA (3-7%) among the 45 unconfirmed cases by MAT, demonstrating the value of PCR in the early diagnosis of human leptospirosis.     

Simultaneous detection and differentiation of pathogenic and nonpathogenic Leptospira spp. by multiplex real-time PCR (TaqMan) assay

Leptospirosis, caused by pathogenic Leptospira, is one of the most important zoonoses in the world. Several molecular techniques have been developed for detection and differentiation between pathogenic and saprophytic Leptospira spp. The aim of this study was to develop a rapid and simple assay for specific detection and differentiation of pathogenic Leptospira spp. by multiplex real-time PCR (TaqMan) assay using primers and probes targeting Leptospira genus specific 16S ribosomal RNA gene, the pathogen specific lig A/B genes and nonpathogen Leptospira biflexa specific 23S ribosomal RNA gene. Sixteen reference strains of Leptospira spp. including pathogenic and nonpathogenic and ten other negative control bacterial strains were used in the study. While the 16S primers amplified target from both pathogenic and non-pathogenic leptospires, the ligA/B and the 23S primers amplified target DNA from pathogenic and non-pathogenic leptospires, respectively. The multiplex real-time PCR (TaqMan) assay detection limit, that is, the sensitivity was found approximately 1 x 10(2) cells/ml for ligA/B gene and 23S ribosomal RNA gene, and 10 cells/ml 16S ribosomal RNA. The reaction efficiencies were 83-105% with decision coefficients of more than 0.99 in all multiplex assays. The multiplex real-time PCR (TaqMan) assay yielded negative results with the ten other control bacteria. In conclusion, the developed multiplex real-time PCR (TaqMan) assay is highly useful for early diagnosis and differentiation between pathogenic and non-pathogenic leptospires in a reaction tube as having high sensitivity and specificity.     

Application of PCR for Specific Diagnosis of Leptospirosis in Humans in Ukraine

Leptospirosis remains one of the most widespread zoonotic diseases in the world and Ukraine, in particular. Ukrainian clinicians have been faced with early detection of the disease due to the availability of only a serological method for routine diagnostics in Ukraine, namely the microscopic agglutination test (MAT). This paper demonstrates the first results of the complex application of MAT and polymerase chain reaction (PCR) for routine verification of leptospirosis, which were first applied simultaneously in Lviv Oblast of Ukraine in 2016. We examined the sera of 150 patients clinically suspected of leptospirosis, 31 of whom were treated at the Lviv Oblast Clinical Hospital for Infectious Diseases (LOCHID). The application of PCR during the first seven days of the disease allowed increasing the share of confirmed leptospirosis cases by 16,1% in patients that were treated in LOCHID during 2016–2017.Key words: Leptospirosis, diagnostics, microagglutination test, polymerase chain reaction     

Flying foxes as carriers of pathogenic Leptospira species

Recent serologic studies have identified flying foxes (Pteropus spp.) as carriers of leptospirosis; however, little is known about the role of flying foxes as carriers of pathogenic Leptospira spp. To determine if Australian Pteropus spp. are carriers of pathogenic Leptospira spp., TaqMan real-time polymerase chain reaction (PCR) was used to detect leptospiral DNA in kidney and urine specimens from four species of flying fox, including the spectacled flying fox (Pteropus conspicillatus), black flying fox (Pteropus alecto), grey-headed flying fox (Pteropus poliocephalus), and little red flying fox (Pteropus scapulatus). Of the 173 kidney samples tested, 19 (11%) were positive for leptospiral DNA. Positive individuals were detected in all four species; significant differences in prevalence were not detected between species, between species within the same geographic area, or between geographically separated samples from the same species. Of the 46 urine samples tested, 18 (39%) tested positive by PCR, confirming that flying foxes shed leptospires into the environment. The detection of leptospiral DNA in the kidneys and urine of flying foxes suggests that flying foxes are carriers of pathogenic Leptospira spp. No evidence collected in the present study, however, suggests that flying foxes pose a significant risk of leptospirosis to the wider community or that humans who are in regular, close contact with flying foxes are at risk for leptospirosis.     

A prospective study to evaluate the accuracy of rapid diagnostic tests for diagnosis of human leptospirosis: Result from THAI-LEPTO AKI study

BackgroundRapid diagnostic tests (RDTs) have become widely used in low-resource settings for leptospirosis diagnostic. This study aims to evaluate the diagnostic performance of the five commercially available RDTs to detect human IgM against Leptospira spp. in Thai population.Methodology/Principal findingsNinety-nine serum samples from Leptospirosis suspicious patients were tested with five RDTs, including Medical Science Public Health, Leptocheck-WB, SD bioline, TRUSTline, and J.Mitra. The case definition was based on MAT, qPCR, and culture results. Diagnostic accuracy was determined based on the first day of enrollment in an overall analysis and stratified according to days post-onset of fever. The five RDTs had overall sensitivity ranging from 1.8% to 75% and specificity ranging from 52.3% to 97.7%. Leptocheck-WB had high sensitivity of 75.0%. The sensitivity of five RDTs increased on days 4–6 post-onset of fever, while the specificity of all tests remained relatively stable at different days post-onset of fever.Conclusions/SignificanceThe tested RDTs showed low sensitivity. Therefore, based on the present study, five commercially available RDTs might not be an appropriate test for acute leptospirosis screening in the Thai population.     

Molecular diagnosis of eosinophilic meningitis due to Angiostrongylus cantonensis (Nematoda: Metastrongyloidea) by polymerase chain reaction-DNA sequencing of cerebrospinal fluids of patients

Cerebrospinal fluid (CSF) samples from clinically diagnosed patients with detectable Angiostrongylus canto-nensis-specific antibodies (n = 10), patients with clinically suspected cases that tested negative for A. cantonensis-an-tibodies (n = 5) and patients with cerebral gnathostomiasis (n = 2) and neurocysticercosis (n = 2) were examined by a single-step polymerase chain reaction (PCR) method using the AC primers for the 66-kDa native protein gene. The PCR method detected A. cantonensis DNA in CSF samples from four of 10 serologically confirmed angiostrongyliasis cases. The PCR results were negative for the remaining CSF samples. The nucleotide sequences of three positive CSF-PCR samples shared 98.8-99.2% similarity with the reference sequence of A. cantonensis. These results indicate the potential application of this PCR assay with clinical CSF samples for additional support in the confirmation of eosinophilic meningitis due to A. cantonensis.     

Lipopolysaccharide Specific Immunochromatography Based Lateral Flow Assay for Serogroup Specific Diagnosis of Leptospirosis in India

Background

Leptospirosis is a re-emerging infectious disease that is under-recognized due to low-sensitivity and cumbersome serological tests. MAT is the gold standard test and it is the only serogroup specific test used till date. Rapid reliable alternative serogroup specific tests are needed for surveillance studies to identify locally circulating serogroups in the study area.

Methods/Principal Findings

In the present investigation the serological specificity of leptospiral lipopolysaccharides (LPS) was evaluated by enzyme linked immunosorbent assay (ELISA), dot blot assay and rapid immunochromatography based lateral flow assay (ICG-LFA). Sera samples from 120 MAT positive cases, 174 cases with febrile illness other than leptospirosis, and 121 seronegative healthy controls were evaluated for the diagnostic sensitivity and specificity of the developed assays. LPS was extracted from five locally predominant circulating serogroups including: Australis (27.5%), Autumnalis (11.7%), Ballum (25.8%), Grippotyphosa (12.5%), Pomona (10%) and were used as antigens in the diagnostics to detect IgM antibodies in patients’ sera. The sensitivity observed by IgM ELISA and dot blot assay using various leptospiral LPS was >90% for homologous sera. Except for Ballum LPS, no other LPS showed cross-reactivity to heterologous sera. An attempt was made to develop LPS based ICG-LFA for rapid and sensitive serogroup specific diagnostics of leptospirosis. The developed ICG-LFA showed sensitivity in the range between 93 and 100% for homologous sera. The Wilcoxon analysis showed LPS based ICG-LFA did not differ significantly from the gold standard MAT (P>0.05).

Conclusion

The application of single array of LPS for serogroup specific diagnosis is first of its kind. The developed assay could potentially be evaluated and employed for as MAT alternative.     

The evolution of Pyrosequencing® for microbiology: From genes to genomes

Leptospirosis is caused by Leptospira, gram negative spirochaetes whose microbiologic identification is difficult due to their low rate of growth and metabolic activity. In Colombia leptospirosis diagnosis is achieved by serological techniques without unified criteria for what positive titers are. In this study we compared polymerase chain reaction (PCR) with microbiological culture and dark field microscopy for the diagnosis of leptospirosis. Microbiological and molecular techniques were performed on 83 samples of urine taken from bovines in the savannahs surrounding Bogotá in Colombia, with presumptive diagnosis of leptospirosis. 117 samples of urine taken from healthy bovines were used as negative controls. 83 samples were MAT positive with titers ≥ 1:50; 81 with titers ≥ 1:100; and 66 with titers ≥ 1:200. 36% of the total samples (73/200) were Leptospira positives by microbiological culture, 32% (63/200) by dark field microscopy and 37% (74/200) by PCR. Amplicons obtained by PCR were 482 base pair long which are Leptospira specific. An amplicon of 262 base pairs typical of pathogenic Leptospira was observed in 71 out of the 74 PCR positive samples. The remaining 3 samples showed a 240 base pair amplicon which is typical of saprophytic Leptospira. PCR as a Leptospira diagnosis technique was 100% sensitive and 99% specific in comparison to microbiological culture. Kappa value of 0.99 indicated an excellent concordance between these techniques. Sensitivity and specificity reported for MAT when compared to microbiological culture was 0.95 and 0.89 with a ≥ 1:50 cut off. PCR was a reliable method for the rapid and precise diagnosis of leptospirosis when compared to traditional techniques in our study. The research presented here will be helpful to improve diagnosis and control of leptospirosis in Colombia and other endemic countries.     

Rapid diagnosis of leptospirosis in patients with different clinical manifestations by 16S rRNA gene based nested PCR

Leptospirosis, a zoonosis of global importance and it is underreported in India and more than 50,000 severe cases are reported each year. Here we present the evaluation of 16S rRNA based nested PCR assay for the rapid identification of human leptospires using serum and urine samples. The study includes 261 suspected cases for leptospirosis with different clinical manifestations. 16S rRNA based nested PCR assay was compared and evaluated against the conventional serological methods such as MAT and ELISA. The technique enabled amplification of a 289 bp product with notable percentage of positivity in all sample groups including 94.8 in pediatric cases, 93 in pregnant women, 94.2 in renal failure, 87.8 in jaundice and 94.6 in common febrile cases. The sensitivity and specificity was 94.4% and 100%, respectively. The technique proved to be prompt and effective for the diagnosis of leptospiral infection at the acute phase of the disease. PCR based approach detects leptospiral DNA from the clinical samples both at the acute and leptospiruria phase on comparison with its counter parts where detection is made possible only after 7 days or 7–30 days post-infection. In this regard PCR based diagnosis of leptospirosis should be made available for clinicians for the early diagnosis and prompt treatment of the disease.     

Employment of broad range 16S rDNA PCR and sequencing in the detection of aetiological agents of meningitis

To employ partial 16S rDNA PCR and automated sequencing technique to identify non-culturable causal agents of bacterial meningitis, 73 peripheral blood samples and 413 culture-negative and eight culture-positive CSF clinical specimens from patients with suspected acute meningitis were examined for the presence of bacterial genomic DNA employing broad range 16S rDNA PCR followed by sequencing of the amplicons. In blood samples, 63/73 specimens were PCR positive (86.3%) and after direct sequencing of the PCR amplicons, only 12.7% (8/63) gave clear sequencing results and 55/63 (87.3%) were mixed with more than one organism detected. The mixed PCR amplicons were separated by using PAGE and mixed amplicons from 29/55 (52.7%) specimens were successfully identified through sequencing. Of the CSF samples, 8/8 culture-positive samples were also PCR positive and 45/413 (10.9%) of culture-negative gave a strong PCR signal and 88/413 (21.3%) specimens yielded a weak PCR signal. The remaining 280 culture-negative specimens were also PCR negative. Nested PCR was set up for the 88 weak positive samples and yielded 72/88 (81.8%) strong positives, with the remainder failing to amplify 133/413 (32.2%) culture-negative samples were PCR positive. In this study, the most common bacteria identified from blood specimens were Neisseria meningitidis, 13/63 (20.6%); Streptococcus spp, 5/63 (7.9%); Acinetobacter spp and Pseudomonas spp 4/63 (6.3%). From culture-negative CSF, the pattern was different in that Staphylococcus spp (13/58, 22.4%), Neisseria meningitidis (9/58, 15.52%) and Pseudomonas spp (8/58, 14.79%), were the most frequent. Overall, 16S rRNA broad-range PCR combined with direct DNA sequencing is a valuable molecular tool to aid with the detection as well as identification of non-culturable aetiological agents of acute bacterial meningitis and can augment cultural methods in the diagnosis of central nervous system infections in patients who have been treated with antibiotics. However, this study demonstrates that contamination is an important complication of the molecular assay, which should be attempted to be eliminated through careful laboratory controls. Hence there should be careful interpretation of any molecular finding, in tandem with other laboratory findings, such as culture, immunological and biochemical markers, and the clinical scenario of the patient.     

Direct PCR assay for detection ofNeisseria meningitidis in human cerebrospinal fluid

A PCR amplification was performed to detectNeisseria meningitidis insertion sequence1106 (IS-1106) in the humancerebrospinalfluid (CSF) in cases of meningitis. The study included 27 CSF samples from suspected meningitis patients. Although the inflammatory response in most of the samples was slightly increased, the results showed that 7 (26%) and 8 (30%) CSF samples were diagnosed as meningococcal meningitis by Gram staining and by culture, respectively. The primers of theIS-1106 were used for direct diagnosis ofN. meningitidis in the human spinal fluid after a minor treatment of the CSF samples. The sample was diagnosed as meningococcal meningitis, if a DNA band of about 600 bp was detected in the ethidium bromide-stained agarose gel. The 27 CSF samples were analyzed in a random manner. Of these, 18 samples including the Gram staining- and culture-positive samples were also positive in PCR amplification. However, a CSF sample, which was diagnosed to be meningococcal meningitis in culture was negative in both Gram staining and PCR analysis. The specificity of theIS-1106 primers was determined to be 95%, with 100% sensitivity in comparison to Gram staining and culture. The primers were sensitive to 10 pg or more of meningococcal DNA. In addition, the PCR amplification showed high predictive values (89 and 100%) in diagnosing meningitis in patients that were negative and positive responders when tested by culture and by Gram staining. In conclusion, the PCR amplification ofIS-1106 ofN. meningitidis is specific and sensitive to both culture-positive and-negative meningococcal meningitis. Hence, PCR assay is highly recommended for use in a rapid diagnosis of suspected meningitis patients.     

Sero-prevalence of anti-Leptospira antibodies and associated risk factors in rural Rwanda: A cross-sectional study

BackgroundLeptospirosis is a zoonotic disease transmitted through the urine of wild and domestic animals, and is responsible for over 50,000 deaths each year. In East Africa, prevalence varies greatly, from as low as 7% in Kenya to 37% in Somalia. Transmission epidemiology also varies around the world, with research in Nicaragua showing that rodents are the most clinically important, while studies in Egypt and Chile suggest that dogs may play a more important role. There are no published studies of leptospirosis in Rwanda.Methods & findingsWe performed a cross-sectional survey of asymptomatic adults recruited from five occupational categories. Serum samples were tested using ELISA and Microscopic Agglutination Test (MAT). We found that 40.1% (151/377) of asymptomatic adults had been exposed to Leptospira spp. Almost 36.3% of positive subjects reported contact with rats (137/377) which represent 90.7% among positive leptospira serology compared with 48.2% of negative subjects (182/377) which represent 80.5% among negative leptospira serology (OR 2.37, CI 1.25–4.49) and 1.7 fold on prevalence ratio and 2.37 of odd ratio. Furthermore, being a crop farmer was significantly associated with leptospirosis (OR 2.06, CI 1.29–3.28). We identified 6 asymptomatic subjects (1.6%) who met criteria for acute infection.ConclusionsThis study demonstrates a high prevalence of leptospiral antibodies infection among asymptomatic adults in rural Rwanda, particularly relative to neighboring countries. Although positive subjects were more likely to report rat contact, we found no independent association between rats and leptospirosis infection. Nonetheless, exposure was high among crop farmers, which is supportive of the hypothesis that rats together with domestic livestock might contribute to the transmission.Further studies are needed to understand infecting Leptospira servers and elucidate the transmission epidemiology in Rwanda and identify means of host transmitters.     

Expanding the Diagnostic Use of PCR in Leptospirosis: Improved Method for DNA Extraction from Blood Cultures

Background

Leptospirosis is a neglected zoonosis of ubiquitous distribution. Symptoms are often non-specific and may range from flu-like symptoms to multi-organ failure. Diagnosis can only be made by specific diagnostic tests like serology and PCR. In non-endemic countries, leptospirosis is often not suspected before antibiotic treatment has been initiated and consequently, relevant samples for diagnostic PCR are difficult to obtain. Blood cultures are obtained from most hospitalized patients before antibiotic therapy and incubated for at least five days, thus providing an important source of blood for PCR diagnosis. However, blood cultures contain inhibitors of PCR that are not readily removed by most DNA-extraction methods, primarily sodium polyanetholesulfonate (SPS).

Methodology/Principal Findings

In this study, two improved DNA extraction methods for use with blood cultures are presented and found to be superior in recovering DNA of Leptospira interrogans when compared with three previously described methods. The improved methods were easy and robust in use with all tested brands of blood culture media. Applied to 96 blood cultures obtained from 36 patients suspected of leptospirosis, all seven patients with positive convalescence serology were found positive by PCR if at least one anaerobic and one aerobic blood culture, sampled before antibiotic therapy were tested.

Conclusions/Significance

This study suggests that a specific and early diagnosis can be obtained in most cases of severe leptospirosis for up to five days after initiation of antimicrobial therapy, if PCR is applied to blood cultures already sampled as a routine procedure in most septic patients.