Multiplex real-time PCR assay for detection of Entamoeba histolytica, Giardia intestinalis, and Cryptosporidium spp

Entamoeba histolytica, Giardia intestinalis, and Cryptosporidium spp. are not only three of the most important and common diarrhea-causing parasitic protozoa, but they often have similar clinical presentations. Microscopic diagnosis of these parasites is neither sensitive nor specific. Recently, more specific and sensitive alternative molecular methods (polymerase chain reaction [PCR] and antigen detection tests) have been introduced for all three of these parasitic infections. The use of these molecular diagnostic tests in routine diagnostic laboratories is still limited. In this study, we developed a multiplex real-time PCR assay for the simultaneous detection of E. histolytica, G. intestinalis, and Cryptosporidium spp. in one reaction using species-specific probes. This assay was evaluated using clinical specimens and was found to be quite sensitive and specific. The reagents used in this multiplex PCR assay can also be used for detection of these parasites individually. The use of this real-time PCR multiplex assay in developing countries at present will have limited scope for routine diagnosis because the cost will be high for a single test, although in the developed world, the test could see immediate application.     

A pentaplex real-time polymerase chain reaction assay for detection of four species of soil-transmitted helminths

Soil-transmitted helminth infections remain a major public health burden in low- and middle-income countries. The traditional diagnosis by microscopic examination of fecal samples is insensitive and time-consuming. In this study, a pentaplex real-time polymerase chain reaction (PCR) was evaluated for the simultaneous detection of Ancylostoma, Necator americanus, Ascaris lumbricoides, and Strongyloides stercoralis. The results were compared with those obtained by conventional parasitological diagnostic methods. Real-time PCR was positive in 48 of 77 samples (62.3%) and microscopic examination was positive in six samples (7.8%) only (P < 0.05). In conclusion, the real-time PCR assay described in this study provides a specific and sensitive diagnostic tool for the detection of these four helminth species in epidemiological studies and monitoring of treatment programs.     

High throughput multiplex PCR and probe-based detection with Luminex beads for seven intestinal parasites

Polymerase chain reaction (PCR) assays for intestinal parasites are increasingly being used on fecal DNA samples for enhanced specificity and sensitivity of detection. Comparison of these tests against microscopy and copro-antigen detection has been favorable, and substitution of PCR-based assays for the ova and parasite stool examination is a foreseeable goal for the near future. One challenge is the diverse list of protozoan and helminth parasites. Several existing real-time PCR assays for the major intestinal parasites-Cryptosporidium spp., Giardia intestinalis, Entamoeba histolytica, Ancylostoma duodenale, Ascaris lumbricoides, Necator americanus, and Strongyloides stercoralis-were adapted into a high throughput protocol. The assay involves two multiplex PCR reactions, one with specific primers for the protozoa and one with specific primers for the helminths, after which PCR products are hybridized to beads linked to internal oligonucleotide probes and detected on a Luminex platform. When compared with the parent multiplex real-time PCR assays, this multiplex PCR-bead assay afforded between 83% and 100% sensitivity and specificity on a total of 319 clinical specimens. In conclusion, this multiplex PCR-bead protocol provides a sensitive diagnostic screen for a large panel of intestinal parasites.     

改良加藤厚涂片法和PCR法检测华支睾吸虫感染的 效果比较

目的　比较改良加藤厚涂片法（Kato?Katz法）和PCR法对现场人群粪便样本中华支睾吸虫感染的检出率,为华支睾吸虫感染检测方法的选择提供依据。方法　在2016年广西壮族自治区藤县人体华支睾吸虫感染流行病学调查的基础上,随机抽取133份粪便样本,-20 ℃保存。分别采用Kato?Katz法（1粪3检）和PCR法检测华支睾吸虫感染,比较两种方法对华支睾吸虫感染的检出率,采用Kappa分析评价两种检测方法结果的一致性。结果　133份人群粪便样本中,华支睾吸虫感染总检出率为77.44%（103/133）,其中Kato?Katz法检出率为57.14%（76/133）,PCR法检出率为70.68%（93/133）, PCR法检出率显著高于Kato?Katz法（[χ2] = 26.15,P <0.01）。76份Kato?Katz法阳性粪便样本中,88.16%（67/76）的样本PCR法扩增阳性;57份Kato?Katz法阴性样本中,47.37%（27/57）的样本PCR法扩增阳性。PCR法对每g粪便虫卵数（EPG）> 1 000的粪便样本中华支睾吸虫感染的检出率（94.7%,18/19）高于EPG < 1 000的样本（85.96%,49/57）,但差异无统计学意义([χ2] = 1.05,P = 0.436)。两种方法检出率一致性检验结果一般（Kappa = 0.73）。结论　PCR法对人群华支睾吸虫感染的检出率显著高于Kato?Katz法。建议在华支睾吸虫感染度较轻的地区,采用Kato?Katz法结合PCR法进行检测,以提高检出率。     

应用多重PCR法鉴定蚊胃血血源的研揪

目的建立多重PCR法鉴定蚊胃血来源。方法依据常见蚊吸血对象(人、牛、猪和犬)的线粒体DNA细胞色素b序列的差异,设计种特异引物,建立多重PCR法,并应用该法检测现场按蚊标本。结果应用多重PCR法共检测249只按蚊,血源来自牛和猪的共91只和63只,未检出吸人血按蚊。立即处死并干燥保存的现场按蚊标本检测成功率最高,为92.50%。结论多重PCR法鉴定蚊胃血血源快速、灵敏,结果客观、可靠。     

Development of multiplex polymerase chain reaction for detection of feline hemotropic mycoplasma in blood and tissue specimens

A multiplex polymerase chain reaction (PCR) was developed for the detection of feline hemotropic mycoplasmas which simultaneously differentiates infections of Mycoplasma haemofelis (Mhf), Candidatus Mycoplasma haemominutum (CMhm) and Candidatus Mycoplasma turicensis (CMtc) in feline blood and spleen. These organisms are responsible for the cause of various pathogenicity of feline infectious anemia. These infections are difficult to be detected by microscopic examination, the most commonly used method for general laboratory diagnoses. Specific primers were designed by selected consensus 16S rDNA sequences of three distinct species. The multiplex PCR assay developed in this study was sensitive and specific with detection limit 100 copies/microl DNA of Mhf and CMhm and 10 copies/microl DNA of CMtc. No amplicons could be amplified for other blood parasites or bacterial pathogens. This multiplex PCR will allow studies of pathogenicity and the monitoring of clinical treatment.     

Polymerase chain reaction-based differential diagnosis of Ancylostoma duodenale and Necator americanus infections in humans in northern Ghana

We evaluated a two-step semi-nested polymerase chain reaction (PCR)-based approach for the specific detection of Ancylostoma duodenale DNA in human faeces. The test was used to determine to what extent this species of hookworm is present in the regions of Bolgatanga and Garu of northern Ghana. Initially, the sensitivity and specificity of the PCR were tested using a range of well-defined control samples. Subsequently, a total of 378 human faecal DNA samples from Bolgatanga and Garu were subjected to the PCR. The results were compared with those obtained using a previously established PCR for the specific detection of Necator americanus DNA in human faeces. Infection with A. duodenale was recorded in 74 (19.6%) samples and N. americanus in 278 (73.5%), of which 64 (16.9%), represented co-infections with both species. While A. duodenale was predominantly detected in the samples from Bolgatanga, infections in Garu related almost exclusively to N. americanus. The results showed that the present PCR approach is a valuable complementary tool for the diagnosis of A. duodenale infection in humans in Ghana, having implications for epidemiological studies and for the monitoring of the success of control programmes in regions in Africa.     

Identification of Pseudomonas aeruginosa, Burkholderia cepacia complex, and Stenotrophomonas maltophilia in respiratory samples from cystic fibrosis patients using multiplex PCR

A multiplex PCR method was developed to identify P. aeruginosa, B. cepacia complex, and S. maltophilia directly in sputum and oropharyngeal samples from CF patients. One hundred and six patients (53 male, and 53 female) attending our pulmonology clinic were studied from September 2000-April 2001. Two hundred and fifty-seven samples were cultured in selective media and submitted to multiplex PCR reactions, using three primer pairs targeting specific genomic sequences of each species, with an additional primer pair targeting a stretch of ribosomal 16S DNA, universal for bacteria, to act as a control. P. aeruginosa was isolated by culture in 56% of samples, B. cepacia complex in 4.3%, and S. maltophilia in 2.7%, while multiplex PCR identified P. aeruginosa in 78.7%, B. cepacia complex in 3.9%, and S. maltophilia in 3.1% of samples. Multiplex PCR results were verified by PCR reactions using different species-specific primers described in the literature and DNA sequencing of amplicons from a few samples. Comparing to culture results, the sensitivity and specificity values of multiplex PCR for bacterial identification were, respectively, 97.2% and 45.5% for P. aeruginosa, 45.5% and 97.9% for B. cepacia complex, and 40% and 97.6% for S. maltophilia. All 10 multiplex PCR-positive results for B. cepacia complex were confirmed using other species-specific primers described in the literature, while this approach confirmed results for S. maltophilia identification in 7/8 samples (87.5%). Sequencing of amplicons from samples culture-negative but multiplex PCR-positive for P. aeruginosa and B. cepacia complex confirmed their identity, while minor nucleotide differences among amplicons ruled out the hypothesis of PCR contamination.     

Application of real-time polymerase chain reaction (PCR) analysis for detection and discrimination of malaria parasite species in Thai patients

A TaqMan real-time PCR system was used to detect and discriminate the 4 species of human malaria parasites in clinical blood samples. A 150-base pair (bp) region of the small subunit ribosomal RNA (SSU rRNA) gene of each malaria parasite, including species-specific sequences to be detected by TaqMan probe, was used as a target for PCR analysis. The PCR method used universal primers and species-specific TaqMan probes for Plasmodium falciparum, P. vivax, P. ovale, and P. malariae. The detection threshold for the method, as determined with serial dilution of cultured P. falciparum-infected erythrocytes, was 5 parasite-infected erythrocytes per reaction. Fifty blood samples of falciparum malaria and a second set of 50 samples of vivax malaria, diagnosed by microscopic examination at the Hospital for Tropical Diseases, Mahidol University, Thailand, were analyzed by real-time PCR. In the 50 samples of microscopically-diagnosed falciparum malaria, 40 were regarded as P. falciparum single infection, 7 were P. falciparum and P. vivax mixed infections, and 3 were P. vivax single infection by real-time PCR. In the second set of 50 samples of microscopically diagnosed vivax malaria, all were considered P. vivax single infection by PCR. Neither P. ovale nor P. malariae infection was identified in the 100 blood samples. Real-time PCR analysis was shown to be more sensitive and accurate than routine diagnostic methods. Application and extension of the PCR method reported here will provide a powerful tool for further studies of malaria.     

Comparison of three multiplex PCR assays for the detection of respiratory viral infections: evaluation of xTAG respiratory virus panel fast assay, RespiFinder 19 assay and RespiFinder SMART 22 assay

ABSTRACT: BACKGROUND: A broad spectrum of pathogens is causative for respiratory tract infections, but symptoms are mostly similar. Therefore, the identification of the causative viruses and bacteria is only feasible using multiplex PCR or several monoplex PCR tests in parallel. METHODS: The analytical sensitivity of three multiplex PCR assays, RespiFinder-19, RespiFinder- SMART-22 and xTAG-Respiratory-Virus-Panel-Fast-Assay (RVP), were compared to monoplex real-time PCR with quantified standardized control material. All assays include the most common respiratory pathogens. RESULTS: To compare the analytical sensitivity of the multiplex assays, samples were inoculated with 13 different quantified viruses in the range of 101 to 105 copies/ml. Concordant results were received for rhinovirus, whereas the RVP detected influenzavirus, RSV and hMPV more frequently in low concentrations. The RespiFinder-19 and the RespiFinder-SMART-22 showed a higher analytical sensitivity for adenoviruses and coronaviruses, whereas the RVP was incapable to detect adenovirus and coronavirus in concentrations of 104 copies/ml. The RespiFinder-19 and RespiFinder-SMART-22A did not detect influenzaviruses (104 copies/ml) and RSV (103 copies/ml). The detection of all 13 viruses in one sample was only achieved using monoplex PCR. To analyze possible competitive amplification reactions between the different viruses, samples were further inoculated with only 4 different viruses in one sample. Compared to the detection of 13 viruses in parallel, only a few differences were found. The incidence of respiratory viruses was compared in tracheal secretion (TS) samples (n = 100) of mechanically ventilated patients in winter (n = 50) and summer (n = 50). In winter, respiratory viruses were detected in 32 TS samples (64%) by RespiFinder-19, whereas the detection rate with RVP was only 22%. The most frequent viruses were adenovirus (32%) and PIV-2 (20%). Multiple infections were detected in 16 TS samples (32%) by RespiFinder-19. Fewer infections were found in summer (RespiFinder-19: 20%; RVP: 6%). All positive results were verified using monoplex PCR. CONCLUSIONS: Multiplex PCR tests have a broad spectrum of pathogens to test at a time. Analysis of multiple inoculated samples revealed a different focus of the detected virus types by the three assays. Analysis of clinical samples showed a high concordance of detected viruses by the RespiFinder-19 compared to monoplex tests.     

Identification of human malaria parasites and detection of mixed infection in Thai patients by nested PCR

The species-specific nested PCR previously described by Snounou and others, for detecting the four species of human malaria parasites, is evaluated in the current study testing 40 blood samples from malaria patients admitted during July-September, 2003, at the Hospital for Tropical Diseases, Faculty of Tropical Medicine, Mahidol University, Thailand. Parasite DNA of each blood sample was extracted and purified by QIAamp. DNA mini kit. Nested PCR was performed using genus-specific primers for the first PCR cycle and species-specific primer for the second cycle. Thin and thick smears were also made, stained with Giemsa, and examined by expert microscopists. Only one of 40 samples (2.5%) was identified as Plasmodium malariae infection by both microscopy and nested PCR. Twenty blood samples (50%) were identified as Plasmodium falciparum infections by both methods. However, 19 blood samples (47.5%) were reported as Plasmodium vivax infections by microscopic methods, whereas nested PCR could detect a mixed infection of Plasmodium vivax and Plasmodium falciparum in one sample taken from a young girl with 8 ameboid trophozoites of P. vivax per 200 white blood cells. These results demonstrated that the nested PCR assay surpasses microscopy and also offers a clear advantage in the detection of mixed infections, which is important not only for successful medical treatment, but also for the study of malaria epidemiology.     

Application of a circulating-cathodic-antigen (CCA) strip test and real-time PCR, in comparison with microscopy, for the detection of Schistosoma haematobium in urine samples from Ghana

In the detection of parasitic infection, the traditional methods based on microscopy often have low sensitivity and/or specificity compared with the newer, molecular tests. An assay based on real-time PCR and a reagent strip test for detecting circulating cathodic antigen (CCA) have both now been compared with urine filtration and microscopy, in the detection of Schistosoma haematobium infections. Urine samples, obtained from 74 'cases' in areas of Ghana with endemic S. haematobium and 79 'controls' from non-endemic areas, were each checked using the three methods. With the results of the filtration and microscopy taken as the 'gold standard', real-time PCR was found to be 100% specific and 89% sensitive whereas the CCA strips were 91% specific and 41% sensitive. With the samples found to contain > or =50 eggs/10 ml (indicating relatively intense infections), the sensitivities of the PCR and CCA were higher, at 100% and 62%, respectively. As expected, egg counts were negatively correlated with the number of amplification cycles needed, in the PCR, to give a signal that exceeded the background (r=-0.38; P<0.01). Although the real-time PCR and CCA strip tests are very different, both show promise in the detection of S. haematobium infections. The PCR has optimal specificity and high sensitivity but the specificity of the CCA strips and the sensitivity of both tools could still be improved. A more thorough re-evaluation of the sensitivity and specificity of microscopy and these newer diagnostic methods, with an estimation of the cost-effectiveness of each technique, is recommended.     

Comparison of Three Molecular Methods for the Detection and Speciation of Five Human Plasmodium Species

In this study, three molecular assays (real-time multiplex polymerase chain reaction [PCR], merozoite surface antigen gene [MSP]-multiplex PCR, and the PlasmoNex Multiplex PCR Kit) have been developed for diagnosis of Plasmodium species. In total, 52 microscopy-positive and 20 malaria-negative samples were used in this study. We found that real-time multiplex PCR was the most sensitive for detecting P. falciparum and P. knowlesi. The MSP-multiplex PCR assay and the PlasmoNex Multiplex PCR Kit were equally sensitive for diagnosing P. knowlesi infection, whereas the PlasmoNex Multiplex PCR Kit and real-time multiplex PCR showed similar sensitivity for detecting P. vivax. The three molecular assays displayed 100% specificity for detecting malaria samples. We observed no significant differences between MSP-multiplex PCR and the PlasmoNex multiplex PCR kit (McNemar''s test: P = 0.1489). However, significant differences were observed comparing real-time multiplex PCR with the PlasmoNex Multiplex PCR Kit (McNemar''s test: P = 0.0044) or real-time multiplex PCR with MSP-multiplex PCR (McNemar''s test: P = 0.0012).     

环介导等温扩增法检测人粪样中美洲钩虫的方法建立

目的 建立一种快速、高效的环介导等温扩增方法(loop-mediated isothermal amplification, LAMP)检测粪样中美洲钩虫虫卵。方法 根据美洲钩虫ITS基因序列,设计4条LAMP引物,建立LAMP检测法。利用LAMP法检测日本血吸虫、肝吸虫、鞭虫、猪蛔虫、蛲虫的DNA样本以验证LAMP方法的特异度。以美洲钩虫成虫DNA样本,倍比稀释以评价LAMP法的DNA最低检出量。现场采集55份人粪DNA样本,采用改良加藤法、LAMP法和普通PCR方法同时检测,评价LAMP法的敏感性和特异性。结果 LAMP法可特异性扩增美洲钩虫DNA样本。LAMP法可检测最低DNA浓度为1.2 fg/mL。通过现场样本对LAMP进行评价,LAMP法敏感性相对于改良加藤法和PCR法分别是95.24%和100%。特异性分别是97.06%和100%。结论 建立起以虫卵为检测材料的检测美洲钩虫病的LAMP法,为美洲钩虫病提供特异、敏感、高效的检测方法。     

SYBR Green荧光定量粪检PCR法检测日本血吸虫感染的敏感性研究

目的建立快速、敏感、特异的检测日本血吸虫感染的SYBR Green荧光定量PCR法,准确评估其敏感性。方法将计数的日本血吸虫虫卵掺人健康水牛粪样中,制备人工阳性粪样。采用改良QIAamp DNA Stool Kit粪样DNA提取方法,提取人工阳性粪样DNA,进行SYBR Green荧光定量PCR,建立Ct值与粪样中克粪虫卵数（EPG）的关系;提取单独（不与牛阴性粪样混合）日本血吸虫虫卵DNA与虫卵生理盐水冲洗液DNA,行定量PCR检测,以对本方法进行严格质量控制。结果每200mg粪样仅含1个虫卵时,荧光定量PCR仍呈阳性,人工阳性粪样EPG的对数与PCR的Ct值存在线性关系。结论SYBR Green荧光定量粪检PCR法检测日本血吸虫虫卵DNA敏感性高,EPG对数与PCR的Ct值存在线性关系。     

Scope and limitations of a multiplex conventional PCR for the diagnosis of S. stercoralis and hookworms

ObjectivesDescribe the diagnostic characteristics of a conventional multiplex PCR for the diagnosis of S. stercoralis, N. americanus and Ancylostomas spp.MethodsFecal samples were collected from a cross-sectional study in Orán department, Salta province, Argentina. The stool samples were analyzed using concentration-sedimentation, Harada Mori, McMaster, and Baermann techniques. DNA was extracted from 50 mg fecal sample using the FastPrep® Spin Kit for Soil. Three pairs of primers were used for the amplification of three products of 101, 330, and 577 base pairs (bp) for S. stercoralis, N. americanus and Ancylostoma spp, respectively. The sensitivity and analytical specificity of multiplex PCR were evaluated, as well as the sensitivity and diagnostic specificity, using a composite standard and Bayesian approach.Results and ConclusionsMultiplex PCR did not present cross-reaction with other intestinal parasites, and the detection limit for multiplex PCR was between 2 and 20 pg of genomic DNA. In addition it presented a diagnostic sensitivity of 97.4% for S. stercoralis and 90.3% for hookworms with a specificity of 100% and 87.6%, respectively. PCR identified a higher proportion (p <0.01) of coinfections (15.3%) than microscopic techniques (3.5%). Also, multiplex PCR showed that there was a positive association between S. stercoralis and hookworms (odds ratio = 2.12). However, this association was due to N. americanus (odds ratio= 3.22), since no association was observed between S. stercoralis and Ancylostoma spp. Neither was an association observed between the two species of hookworms.     

牛隐孢子虫多重PCR方法的建立及应用

摘要：目的 为寻求一种牛源隐孢子虫的快速检测手段。方法 根据牛隐孢子虫COWP基因和ITS-1基因,设计合成两对特异性引物,用以特异性扩增牛微小隐孢子虫和安氏隐孢子虫,扩增长度分别为1033bp和263bp。通过反应条件的优化,特异性试验及扩增片段的序列测定的验证,建立多重PCR方法。结果 应用该方法对采集自广西不同地区的1613份牛粪样进行牛隐孢子虫检测,安氏隐孢子虫阳性粪样为189份,微小隐孢子虫阳性粪样1份,与常规隐孢子虫分离鉴定结果一致。结论 表明该方法可用于隐孢子虫的流行病学调查及临床诊断。     

Comparison of direct fecal smear microscopy,culture, and polymerase chain reaction for the detection of Blastocystis sp. in human stool samples

ObjectiveTo compare the sensitivity and specificity of direct fecal smear microscopy, culture, and polymerase chain reaction in the detection of Blastocystis sp. in human stool.MethodsHuman stool samples were collected from a community in San Isidro, Rodriguez, Rizal, Philippines. These samples were subjected to direct fecal smear microscopy, culture and polymerase chain reaction to detect the presence of Blastocystis sp.ResultsOf the 110 stool samples collected, 28 (25%) were detected positive for the presence of Blastocystis sp. by two or more tests. Culture method detected the highest number of Blastocystis-positive stool samples (n=36), followed by PCR of DNA extracted from culture (n=26), PCR of DNA extracted from stool (n=10), and direct fecal smear (n=9). Compared to culture, the sensitivity of the other detection methods were 66.7% for PCR from culture and 19.4% for both PCR from stool and direct fecal smear. Specificity of the methods was high, with PCR from culture and direct fecal smear having 97.3%, while PCR from stool at 95.9%.ConclusionsIn this study, in vitro culture is the best method for detecting Blastocystis sp. in human stool samples.     

Natural Plasmodium infections in Brazilian wild monkeys: reservoirs for human infections?

Four hundred and forty-eight samples of total blood from wild monkeys living in areas where human autochthonous malaria cases have been reported were screened for the presence of Plasmodium using microscopy and PCR analysis. Samples came from the following distinct ecological areas of Brazil: Atlantic forest (N=140), semideciduous Atlantic forest (N=257) and Cerrado (a savannah-like habitat) (N=51). Thick and thin blood smears of each specimen were examined and Plasmodium infection was screened by multiplex polymerase chain reaction (multiplex PCR). The frequency of Plasmodium infections detected by PCR in Alouatta guariba clamitans in the S?o Paulo Atlantic forest was 11.3% or 8/71 (5.6% for Plasmodium malariae and 5.6% for Plasmodium vivax) and one specimen was positive for Plasmodium falciparum (1.4%); Callithrix sp. (N=30) and Cebus apella (N=39) specimens were negative by PCR tests. Microscopy analysis was negative for all specimens from the Atlantic forest. The positivity rate for Alouatta caraya from semideciduous Atlantic forest was 6.8% (16/235) in the PCR tests (5.5, 0.8 and 0.4% for P. malariae, P. falciparum and P. vivax, respectively), while C. apella specimens were negative. Parasitological examination of the samples using thick smears revealed Plasmodium sp. infections in only seven specimens, which had few parasites (3.0%). Monkeys from the Cerrado (a savannah-like habitat) (42 specimens of A. caraya, 5 of Callithrix jacchus and 4 of C. apella) were negative in both tests. The parasitological prevalence of P. vivax and P. malariae in wild monkeys from Atlantic forest and semideciduous Atlantic forest and the finding of a positive result for P. falciparum in Alouatta from both types of forest support the hypothesis that monkeys belonging to this genus could be a potential reservoir. Furthermore, these findings raise the question of the relationship between simian and autochthonous human malaria in extra-Amazonian regions.     

Clinical Significance of Fecal Lactoferrin and Multiplex Polymerase Chain Reaction in Patients with Acute Diarrhea

Background/AimsThe diagnostic yield of fecal leukocyte and stool cultures is unsatisfactory in patients with acute diarrhea. This study was performed to evaluate the clinical significance of the fecal lactoferrin test and fecal multiplex polymerase chain reaction (PCR) in patients with acute diarrhea.MethodsClinical parameters and laboratory findings, including fecal leukocytes, fecal lactoferrin, stool cultures and stool multiplex PCR for bacteria and viruses, were evaluated prospectively for patients who were hospitalized due to acute diarrhea.ResultsA total of 54 patients were included (male, 23; median age, 42.5 years). Fecal leukocytes and fecal lactoferrin were positive in 33 (61.1%) and 14 (25.4%) patients, respectively. Among the 31 patients who were available for fecal pathogen evaluation, fecal multiplex PCR detected bacterial pathogens in 21 patients, whereas conventional stool cultures were positive in only one patient (67.7% vs 3.2%, p=0.000). Positive fecal lactoferrin was associated with presence of moderate to severe dehydration and detection of bacterial pathogens by multiplex PCR (21.4% vs 2.5%, p=0.049; 100% vs 56.5%, p=0.032, respectively).ConclusionsFecal lactoferrin is a useful marker for more severe dehydration and bacterial etiology in patients with acute diarrhea. Fecal multiplex PCR can detect more causative organisms than conventional stool cultures in patients with acute diarrhea.