Introduction of a rapid dipstick assay for the detection of Leptospira-specific immunoglobulin m antibodies in the laboratory diagnosis of leptospirosis in a hospital in Makassar, Indonesia

An easy, rapid and robust dipstick assay for detection of leptospira-specific immunoglobulin M (IgM) antibodies was evaluated on 403 patients admitted for hospitalization because of fever. The clinical symptoms and signs of 35 patients were consistent with leptospirosis. The final diagnosis for the remaining patients was as follows: 136 with typhoid fever, 82 with hepatitis, 74 with malaria, 48 with infections of the respiratory tract, and 20 with fever of unknown origin. The clinical diagnosis of leptospirosis was confirmed for 24 (68.6%) patients by the combined results of the microscopic agglutination test (MAT), the reference test for leptospirosis, and of IgM ELISA, a standard laboratory test for the serodiagnosis of leptospirosis. In addition, serum specimens from 8 (2.2%) patients with a final clinical diagnosis other than leptospirosis were found to be positive in MAT and/or IgM ELISA. Compared with the results of MAT and IgM ELISA a sensitivity of 91.6% and specificity of 93.6% was calculated for the dipstick assay. Most of the serum samples from the laboratory confirmed patients gave a moderate to strong staining intensity of the antigen band of the dipstick and were easy to read. The results demonstrate that the dipstick assay is convenient to use and allows the rapid and accurate confirmation of patients with clinical suspicion of leptospirosis in areas where the disease is endemic.     

Antibody response in typhoid fever in endemic Indonesia and the relevance of serology and culture to diagnosis

Culture and serology were performed on blood and serum samples collected at or shortly after admission from 473 patients presented with suspected clinical typhoid. Clinical symptoms at first presentation including confusion, hepatomegaly, splenomegaly, abdominal pain, anemia, and gastrointestinal bleeding were non-specific as they were observed even more often in non-typhoid patients. Culture confirmed the diagnosis in 65.3% of the patients with typhoid fever as the final diagnosis. The sensitivity (58%) and specificity (98.1%) of a rapid dipstick assay for the detection of S. typhi-specific immunoglobulin M were somewhat lower than those of culture but higher than those of the Widal test. The dipstick assay thus may well be used in the serodiagnosis of typhoid in situation where culture facilities are not available. Combination of test results of dipstick and culture improved sensitivity to 82.5%. In laboratories that perform blood culture the dipstick assay may be used as a rapid screening tests to facilitate a rapid diagnosis. Sensitivity of the dipstick assay strongly increased with duration of illness and was higher for culture positive than for culture negative patients. Duration of illness, and different pathogen and host factors including dose of infection, pathogenicity and antigenicity, and prior antibiotic use are likely to influence the immune response, therefore the result of the dipstick assay. Duration of illness and presence of S. typhi in the blood are major factors that determine severity of disease.     

International multi-centre evaluation of a dipstick assay for human leptospirosis

A dipstick assay for the detection of Leptospira-specific immunoglobulin M (IgM) antibodies in human sera was evaluated in 27 laboratories in 23 countries. 873 serum samples from 711 patients including 329 laboratory-confirmed leptospirosis case patients, 239 noncase patients and 69 patients with viral infections causing heamorrhagic fever were tested. Relative to the results of the reference leptospirosis test, the sensitivity of the dipstick assay was 84.5% for serum samples collected during the first 10 days of the disease and 92.1% for serum samples collected 10-30 days after the onset of disease. The specificity was 87.5% and 94.4%, respectively. Similar to viral haemorrhagic fevers, leptospirosis may cause bleeding. A small number of serum samples from patients with haemorrhagic viral infections gave a weak (1 +) stain. All other samples were negative. In conclusion, the dipstick assay is sensitive and specific and reacts well with serum samples from patients infected with a range of leptospiral strains. It is also easy to use and does not require special equipment or refrigeration. Therefore the assay is ideal for use in developing countries and rural settings.     

感染登革病毒后抗体产生规律及应用于暴发疫情的血清学诊断

目的了解感染登革病毒后特异性抗体产生规律,指导基层实验室应用血清抗体检测方法对暴发疫情的早期病例作出正确诊断,为控制暴发提供实验室依据。方法采集福建省1次暴发登革热疫情中的发热病人和密切接触者血清标本441份,以及恢复期血清22份,用捕获法ELISA检测登革IgG和IgM抗体,并结合病例的流行病学资料进行分析。结果根据本研究规定的病例判定标准,441例中登革热感染者为228例。其中,IgM抗体检出226例,IgG抗体检出119例。226例IgM抗体阳性标本中,IgG抗体同时阳性的为118份;119例IgG阳性标本中,IgM抗体阴性1例;两者均阴性的标本中用RT-PCR方法检测出1例。22份恢复期血清两类抗体检测均为阳性。绝大部分病例集中在10～70岁,病例的职业分布较广,男女比例为1∶1.53。结论登革病毒感染后特异性IgM抗体在发病早期即可检出,并可维持至少一个半月,但个别病例发病早期IgM可能为阴性;IgG抗体随病程的延长,其检出率逐步增高,至发病后50 d,其检出率可达95%左右。本研究结果抗体产生规律同以往研究结果一致,可为基层实验室开展登革热血清学检测和病例诊断提供参考。     

Detection of specific IgM antibodies for the diagnosis of Mycoplasma pneumoniae infections: a clinical evaluation

The diagnostic value of detection of specific IgM antibodies was analysed in Mycoplasma pneumoniae infections. In a retrospective clinical and serological study, M. pneumoniae IgM antibodies were determined by a mu-capture ELISA using enzyme-labelled antigen. The study group consisted of 91 patients with significantly raised titers in paired sera or a single high titer of complement fixation antibodies. About 40% of the patients had been treated with antibiotics ineffective against M. pneumoniae infections prior to admission to hospital. Treatment with erythromycin or tetracycline was shown to give a shorter period of fever compared to if no or ineffective therapy was given. Specific IgM antibodies were detected in about 80% of sera sampled 9 days or more after onset of symptoms. In sera sampled at 7-8 days after onset IgM antibodies were found in about 40% of the sera but only occasionally in sera sampled earlier. In the age group 0-20 years 88% of the patients developed an IgM response. In the higher ages (greater than 60 years) a significantly lower rate of IgM responders was observed.     

Rapid diagnosis of typhoid fever by enzyme-linked immunosorbent assay detection of Salmonella serotype typhi antigens in urine

We developed and evaluated an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies to capture somatic antigen 9 (O9), flagellar antigen d (Hd), and the Vi capsular polysaccharide antigen (Vi) from the urine of persons with and without typhoid fever. Sequential urine samples were collected from 44 patients with blood culture-confirmed typhoid fever and from two control groups. The first control group included patients with brucellosis (n = 12) and those with clinically diagnosed, non-typhoid, acute, febrile illness (n = 27). The second control group was a sample of healthy volunteer laboratory workers (n = 11). When assessed relative to date of fever onset, sensitivity was highest during the first week for all three antigens: Vi was detected in the urine of nine (100%) patients, O9 in 4 (44%) patients, and Hd in 4 (44%) patients. Sequential testing of two urine samples from the same patient improved test sensitivity. Combined testing for Vi with O9 and Hd produced a trend towards increased sensitivity without compromising specificity. The specificity for Vi exceeded 90% when assessed among both febrile and healthy control subjects, but was only 25% when assessed among patients with brucellosis. Detection of urinary Vi antigen with this ELISA shows promise for the diagnosis of typhoid fever, particularly when used within the first week after fever onset. However, positive reactions for Vi antigen in patients with brucellosis must be understood before urinary Vi antigen detection can be developed further as a useful rapid diagnostic test.     

IFAT检测登革热IgM抗体的初步探讨

目的探讨用间接免疫荧光试验（ＩＦＡＴ）检测登革热ＩｇＭ抗体。方法采集１９９５年７～１１月份广东省登革热Ⅰ型流行期间疑似患者发病１～７ｄ血清６２份,用ＩＦＡＴ检测登革热ＩｇＭ抗体。结果阳性率为２４．１９％（１５／６２）,发病４～５ｄ的阳性率最高为４０．００％以上。ＩｇＭ阳性滴度在１∶１０～１∶４０之间,多数为１∶１０（８／１５）。本法与乙脑血清有交叉反应,与其他非登革热血清无交叉反应。在发病５～６ｄ的患者中可同时检测出登革热的ＩｇＭ和ＩｇＧ抗体。结论发病１～３ｄ的血清可进行病毒分离,４～６ｄ的血清可检测ＩｇＭ抗体,如阴性则再检测ＩｇＧ,以提高登革热的检出率。     

Dengue outbreak in Swat and Mansehra,Pakistan 2013;an epidemiological and diagnostic perspective

Objective:To High light some epidemiological,clinical and diagnostic features of dengue fever during an outbreak and the role of different diagnostic techniques to achieve the highest level of accuracy in results.Methods:Blood samples(n=323) were collected along with epidemiological and clinical data from suspected dengue patients who visited different hospitals in Swat and Mansehra district of Pakistan between May-November 2013 during a dengue outbreak.Samples were tested for the detection of viral nucleic acid by real-lime PCR.non structural protein-1(NS1antigen and IgM antibodies by ELISA.Results:Out of 323 cases with clinical dengue infection,304 were positive by one or more diagnostic parameter:201 samples were positive by real-time PCR,209 were positive by NS1 ELISA and 190 were positive by IgM antibodies.Sensitivities of real-time PCR and NS1 F.LISA were comparable for early diagnosis of dengue virus infection.IgM antibody detection assay was found useful for the diagnosis in the samples collected later than day 5 of onset.Conclusions:The use of real-lime PCR or detection of non stnictural protein NS 1 by ELISA followed by IgM antibodies detection can be recommended for early diagnosis of dengue virus infection with a high level of accuracy.     

An enzyme-linked immunosorbent assay to characterize dengue infections where dengue and Japanese encephalitis co-circulate

The diagnostic sensitivity and specificity of detection of anti-dengue IgM by antibody capture enzyme-linked immunosorbent assay (ELISA) was investigated in dengue infections in a variety of clinical settings. Sera from uninfected controls were uniformly negative. Serial specimens from experimental and natural infections showed that viremia and fever terminated as anti-dengue IgM became detectable. Anti-dengue IgM appeared in most cases by the 3rd afebrile day of illness and declined to undetectable levels after 30-60 days. Assay sensitivity was 78% in admission sera (924/1,183; 95% CI = 75-81%) and 97% in paired sera (1,030/1,062; 95% CI = 96-98%) thus exceeding or matching the performance of the hemagglutination-inhibition assay. Measurement of the anti-dengue IgM to anti-Japanese encephalitis IgM ratio correctly identified all sera from 112 patients with strictly defined Japanese encephalitis and 98% (307/312; 95% CI = 96-99%) of sera from patients whose dengue infections were confirmed by virus isolation. Dengue infections could be classified as primary or secondary by determining the ratio of units of dengue IgM to IgG antibody. We propose that measurement of dengue and Japanese encephalitis IgM and IgG antibodies upon admission and discharge from hospital care should replace the hemagglutination inhibition assay as the standard dengue serologic technique in regions where these 2 viruses co-circulate.     

A retrospective analysis of sera collected by the Hemorrhagic Fever Commission during the Korean Conflict

More than 600 sera from 245 patients with a clinical diagnosis of hemorrhagic fever were preserved by the Hemorrhagic Fever Commission during the Korean Conflict, 1951-1954. These sera were tested for IgM- and IgG-specific antibodies to Hantaan virus by enzyme immunoassay and for hantaviral antigen by immunoassay; one serum from each patient was tested by plaque reduction neutralization using both Hantaan and Seoul viruses. Only 15 patients failed to develop antihantaviral antibodies; most sera contained high titered IgM antibody on admission, and all were IgM-seropositive by day 7 after onset. Attempts to detect hantaviral antigen were unsuccessful. All seropositive patients had highest plaque reduction neutralization titers to Hantaan virus, suggesting that this virus was responsible for the disease seen. These results confirm that hemorrhagic fever of the Korean Conflict was due to Hantaan virus and demonstrate that measurement of specific IgM antibody is the method of choice for diagnosis of acute disease.     

Open study on efficacy and safety of levofloxacin in treatment of uncomplicated typhoid fever

The main objective of this study was to determine the clinical efficacy and safety of levofloxacin in an open setting for typhoid fever cases. Patients with clinical signs and symptoms of typhoid fever without previous antimicrobial treatment admitted to affiliated hospitals of the Faculty of Medicine, University Indonesia were included in this study. Adults, 18 years or above, were screened for any serious underlying conditions, pregnancy or possible complications of typhoid fever before final enrollment. Fifty-three subjects were screened, 48 were enrolled. The final diagnosis of enteric fever was made by positive blood culture, polymerase chain reaction or serology, was obtained in 31 cases, in whom one had a concomitant sinus infection and had to be excluded. Thirty patients (11 males, 19 females) aged between 18-58 years (mean 31.7 years) with a history of fever between 1 and 10 days (mean 6.1 days) showed excellent clinical response, becoming afebrile at an average of 2.43 days (range 1-5 days). Adverse effects noted were nausea in 4 patients, vomiting in one and meteorism in another one, which were all difficult to distinguish from the enteric infection. A pruritic rash occurring in two patients may be related to levofloxacin, and insomnia in another patient may be related. Microbiological clearance was obtained both immediately after treatment and at one month. No carrier states were detected in the cases positive for Salmonella typhi or paratyphi. None of the treated typhoid fever cases experienced a clinical relapse. In this open study of levofloxacin 500 mg/day for one week in treatment of uncomplicated typhoid fever, a 100% clinical efficacy was obtained in 30 patients with minimal adverse reactions warranting more intensive studies for this new indication of an old but well known disease in the developing world.     

Detection of dengue infection by combining the use of an NS1 antigen based assay with antibody detection

We analyzed the utility of a commercial NS1 antigen based ELISA (Panbio Dengue Early ELISA) for detection of dengue infection during the early acute phase and anti-Dengue IgM capture ELISA for detecting dengue infection in patients in dengue endemic settings. A total of 145 serum samples collected from febrile suspected dengue patients were tested for the presence of anti-dengue IgM antibody using IgM antibody Capture ELISA (MAC ELISA) and the presence of dengue virus antigen using PanBio Dengue NS1 Antigen Capture ELISA. Of the 145 patient samples tested, 88 (60.7%) were positive for either NS1 antigen or IgM antibody by MAC ELISA. Dengue NS1 antigen-capture ELISA gave an overall positivity rate of 65.9% (58/88), and IgM ELISA gave an overall positivity rate of 60.2% (53/88). Only NS1 antigen can be used to test during the first two days of fever. MAC ELISAbegins to show positive by the third day of illness and gradually its positivity increases. From Day 3 to Day 7, no significant difference in detection rates was seen between the NS1 assay and MAC ELISA. The NS1 antigen assay may be a useful tool for detecting dengue infection during first few days of fever.     

The diagnostic value of the ELISA-Ty test for the detection of typhoid fever in children.

A study on the reliability of an enzyme linked immunosorbent assay (ELISA) for the detection of typhoid fever, the ELISA-Ty test, was conducted, comprising 44 children suffering from bacteriologically confirmed typhoid fever based on the finding of a positive blood culture for Salmonella typhi, 44 children with fever caused by diseases other than typhoid fever based on the finding of negative culture of blood, urine and stool for S. typhi, and 120 healthy children as controls. This ELISA-Ty test measures the concentration of IgM and of IgG against S. typhi in serum. This test is an indirect ELISA test, based on a method that makes use of a mixture of OMPs (outer membrane proteins) in equal proportion serving as antigen, obtained from different strains of S. typhi which are prevalent locally, peroxidase goat antihuman IgG or IgM (Sigma) as conjugate and orthophenylenediamine (Sigma) as chromogen of the substrate. The result of the test was obtained through the assessment of the end product, using a micro ELISA reader (Behring) at wave length of 490 nm. The data revealed that the mean absorbent values found in children with typhoid fever, for IgM and IgG, were significantly higher (p < 0.05) when compared to those in children with non-typhoid fever as well as to those found in children of the control group. The results of this study confirm that the ELISA-Ty test has a high reliability for the detection of typhoid fever in children, based on the finding of a degree of diagnostic sensitivity as high as 95.45% and 90.91% for respectively IgM and IgG, a diagnostic specificity as high as 93.33% for IgM as well as for IgG, a high diagnostic efficiency (94.32% for IgM and 92.05% for IgG), a high diagnostic positive predictive value of 93.33% for IgM and 93.02% for IgG, high negative predictive values of 95.35% for IgM and 91.11% for IgG for use under clinical as well as under field conditions.     

Dot enzyme immunosorbent assay for the serodiagnosis of typhoid fever.

A nitrocellulose membrane strip dotted with a specific 50 kDa outer membrane protein of Salmonella typhi was applied for the serodiagnosis of typhoid fever. Using horseradish peroxidase conjugated IgM and IgG antibodies with 4-chloronaphthol as substrate, antibodies in typhoid patients were clearly visualised as bluish purple dots while sera from patients with non-typhoid fevers gave negative results. The detection of specific IgM and IgG antibodies in typhoid patients suggest either recent or current infection. Combined with the high specificity, reliability and rapidity of the test, the dot EIA technique provides a simple and useful method for the serodiagnosis of typhoid using a single serum specimen.     

Underrecognition of leptospirosis during a dengue fever outbreak in Hawaii, 2001-2002

During the 10-year period from 1997 through 2006, the reported mean annual incidence rate of leptospirosis in the state of Hawaii was 3.3/100,000 with a range of 22-60 infections reported each year. Because the clinical presentation is highly variable, however, leptospirosis illness is challenging to recognize and may be underdiagnosed. To assess whether the incidence may be substantially higher than reported figures indicate, we retrospectively studied the prevalence of anti-Leptospira IgM antibodies among specimens obtained over a 12-month period (May 2001 to April 2002) from patients presenting with febrile illness during a dengue fever outbreak in Hawaii. Of 1206 patients testing negative or indeterminate for dengue, 54 (4.5%; 95% confidence interval: 3.3%-5.7%) were positive for anti-Leptospira IgM antibodies using a commercially available dipstick enzyme-linked immunosorbent assay (ELISA). The most common clinical symptoms reported by laboratory-positive leptospirosis patients were fever (92%), headache (88%), and myalgia (83%). Three clinical symptoms were significantly less common among persons laboratory positive for leptospirosis when compared with the 122 patients who had been diagnosed with dengue fever during the outbreak: rash (p < 0.0001), chills (p = 0.05), and petechiae (p = 0.0005). Laboratory-positive leptospirosis infections were identified in persons exposed on each of the 5 most populous islands and illness onsets spanned a 10-month period, reflecting an endemic pattern of disease. If added to the figures obtained via routine passive surveillance, the number of leptospirosis infections identified through this study would more than double the annual incidence rate for Hawaii during 2001. These findings indicate that many leptospiral infections in Hawaii go undiagnosed. Physicians should maintain a high index of suspicion for leptospirosis when assessing patients presenting with acute febrile illness among residents and visitors to Hawaii.     

The evaluation of a multiplex PCR-DNA dipstick assay with culture method and EZ Typhi Carrier DNA assay to detect Salmonella Typhi in well water samples

IntroductionSalmonella Typhi is the causative agent for typhoid fever that infects people especially in underdeveloped and developing countries. Even though culture method is the gold standard for detection of this pathogen, there are some limitations such as time consuming and less sensitive that leads to the development of a more sensitive and efficient method such as polymerase chain reaction (PCR).ObjectiveTo evaluate the sensitivity of a multiplex PCR-DNA dipstick assay incomparison with culture method and EZTyphi Carrier DNA assay for the detection of S.Typhi in well water samples.MethodsA total of 580 well water samples were collected and filtered using filter membrane. Bacteria trapped on the membrane was enriched in a selective Selenite F and evaluated using these three methods; (i) Culture method: Bacteria culture was plated on a selective media agar plate and further tested by biochemical tests and serotyping (test was performed within 3 days); (ii) EZTyphi Carrier DNA assay: Extracted DNA from bacteria culture was amplified using two sets of primers targeted for S. Typhi (1238 bp) and internal amplification control (IAC) (810 bp) (3 hours); (iii) Multiplex PCR-DNA dipstick assay: Extracted DNA was amplified using four sets of primers targeted for S. Typhi (70 bp), S. Paratyphi A (93 bp), pan-Salmonella (146 bp) and IAC (123 bp) (2 hours).Results & DiscussionFifteen out of 580 water samples were positive S.Typhi by multiplex PCR-DNA dipstick assay while one sample was positiveS. Typhi by EZTyphi Carrier DNA and culture method. This finding proved that the multiplex PCR-DNA dipstick was superior to culture method and PCR-agarose gel electrophoresis since it has higher positivity compared to the other two methods.ConclusionA rapid and sensitive multiplex PCR-DNA dipstick assay can be used to detect S. Typhi in well water especilally in typhoid endemic area.     

Detection of Japanese encephalitis (JE) virus-specific IgM in cerebrospinal fluid and serum samples from JE patients

Detection of Japanese encephalitis virus (JEV)-specific IgM by IgM-capture enzymed-linked immunosorbent assay (IgM-capture ELISA) has been accepted as the standard for serological diagnosis. In the present study, we analyzed the time course of the positive rate of JEV-specific IgM in serum and cerebrospinal fluid (CSF) specimens from confirmed JE patients. Serum and CSF samples were obtained from 155 JE cases for diagnostic purposes at hospitals in Thailand from 2002 to 2004. The levels of specific IgM were assessed by IgM-capture ELISA in the 171 serum and 156 CSF samples. Anti-JEV IgM was detected in 26 of 44 serum samples collected on days 1-4 of the disease period, in 31 of 44 samples collected on days 5-8, in 23 of 26 samples collected on days 9-12, and in all the samples collected on day 13 or later. Specific IgM was detected in 60 of 66 CSF samples collected on days 1-4 of illness, and in all the CSF samples but one collected on day 7 or later. The results indicate that the detection of JEV-specific IgM in CSF by IgM-capture ELISA is a reliable laboratory diagnostic method for confirmation of JE throughout the disease period, while the detection of IgM in serum samples is a reliable method on day 9 or later.     

Production of specific monoclonal antibodies to Salmonella typhi flagellin and possible application to immunodiagnosis of typhoid fever

Four murine monoclonal antibodies (MAbs) to Salmonella typhi flagellin were produced. These MAbs did not react with eight other enterobacterial strains tested: Salmonella enteritidis, Salmonella typhimurium, Salmonella paratyphi A, Escherichia coli, Shigella flexneri, Shigella sonnei, Yersinia enterocolitica, and Campylobacter jejuni. All four MAbs cross-reacted with Salmonella muenchen flagellin indicating specificity for d antigenic flagellar epitope. One MAb (C4) was selected to develop a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) to detect S. typhi flagellin in serum samples. By use of this assay S. typhi flagellar antigen was detected in 95.5% of serum samples from patients with positive hemoculture for S. typhi, in 93.6% of samples from patients with positive serodiagnosis of typhoid fever, in 26% of samples collected from patients who were initially hemoculture-positive for S. typhi and who had undergone 7-8 d of chemotherapy, in 8.5% of samples from healthy persons from an endemic area, and in no samples from healthy persons from a nonendemic area. The presence of high levels of flagellin antibody titers did not interfere with the antigen detection. The detection of S. typhi flagellar antigen in patient serum may have practical value for rapid diagnosis of typhoid fever.     

Usefulness of nested PCR for the diagnosis of scrub typhus in clinical practice: A prospective study

The aims of this study were to determine the diagnostic accuracy and clinical usefulness of using nested polymerase chain reaction (PCR) for the diagnosis of scrub typhus through a prospective comparison of nested PCR and indirect immunofluorescent antibody assay (IFA). We conducted a multi-center prospective study of patients who were suffering with possible scrub typhus infection. Whole blood samples were collected for PCR testing, and sera were obtained for serology evaluation using the indirect IFA and the passive hemagglutination assay (PHA). We prospectively studied 135 patients with possible scrub typhus. One hundred eighteen patients were confirmed as having scrub typhus, 7 patients were undetermined, and 10 patients were confirmed as having other diseases. The results of nested PCR assay showed a sensitivity of 82.2% and a specificity of 100%. Ninety-six of the 118 patients were positive for IgM on their admission day. Of the 22 patients who were negative for IgM antibody at admission, 19 had positive results for nested PCR of the buffy coat. The nested PCR assay of the buffy coat is useful as a rapid and reliable test for confirming the diagnosis of scrub typhus.