Multiplex polymerase chain reaction detection of Streptococcus pneumoniae and Haemophilus influenzae and their antibiotic resistance in patients with community-acquired pneumonia from southwest Iran

Background This study aimed to evaluate the occurrence of Streptococcus pneumoniae and Haemophilus influenzae in sputum of patients with community-acquired pneumonia (CAP) using culture and multiplex polymerase chain reaction (M-PCR) methods and to survey the antibiotic resistance patterns of aforesaid isolates. Result In total, 23.9 % (n = 22/92) of sputum samples showed positive results in the culture method. S. pneumoniae and H. influenzae were isolated from 15 (16.3 %) and 7 (7.6%) samples, respectively. Using M-PCR, 44 (47.8 %) samples were positive for S. pneumoniae and H. influenzae. Of these, S. pneumoniae and H. influenzae were detected in 33 (35.8%) and 11 (11.9%) of the sputum samples, respectively. The sensitivity, specificity, and accuracy rates of PCR in detection of S. pneumoniae in comparison with culture method were 100, 76.6, and 83.6%, respectively. While, the sensitivity, specificity, and accuracy rates of PCR in detection of H. influenzae in comparison with culture method were 100, 95.3, and 95.8%, respectively. Out of 11 isolates of H. influenzae, two strains confirmed as H. influenzae type b (Hib) and 3 isolates were type f. However, 6 isolates were non-typable. The co-trimoxazole and amoxicillin/clavulanate were the less effective antibiotics against S. pneumonia and H. influenzae, respectively. Ceftriaxone with 13.3% resistance rates was the most effective antibiotic against S. pneumoniae, while, clarithromycin, ceftriaxone, and gentamicin with resistance rates of 28.6% for each one were the most effective chemicals against H. influenzae isolates. Conclusion In this study, the prevalence of S. pneumoniae was more than H. influenzae using culture and M-PCR methods. The M-PCR provided better efficiency in detecting the bacterial agents in CAP patients compared to culture method. This method can improve the early detection of pathogens contributed to CAP. The drug resistant S. pneumoniae and H. influenzae indicated the need to develop a codified monitoring program to prevent further spread of these strains.

1149. Application of a Multiplex Polymerase Chain Reaction Test for Diagnosing Bacterial Enteritis in Children in a Real-Life Clinical Setting

Background Although a bacterial multiplex polymerase chain reaction (mPCR) test should be performed selectively in patients with gastrointestinal symptoms consistent with bacterial enteritis, its usefulness has been evaluated upon stool samples as requested by clinicians, without considering the patients’ gastrointestinal symptoms or clinical diagnoses. This study aimed to determine the subjects to bacterial mPCR testing and to interpret the mPCR test results with considering patients’ clinical symptoms and diagnoses. Methods Medical records of 710 pediatric patients for whom a bacterial mPCR test was performed were retrospectively reviewed. Clinical characteristics and mPCR test results were compared between patients with positive mPCR test results (n = 199) and those with negative mPCR test results (n = 511) and between patients in whom inflammatory pathogens (Campylobacter spp. and Salmonella spp.) were identified (n = 95) and those in whom toxigenic pathogens (Clostridium spp.) were identified (n = 70). Results A positive mPCR test result was significantly associated with an older age (p < 0.001), diagnosis of acute gastroenteritis (p = 0.021), presence of hematochezia (p < 0.001), and absence of cough (p = 0.004). The diagnosis of acute gastroenteritis (p = 0.003), presence of fever (p = 0.027) and diarrhea (p = 0.043), and a higher C-reactive protein level (p = 0.025) were significantly associated with the identification of inflammatory pathogens rather than toxigenic pathogens in patients with positive mPCR test results. Conclusion Bacterial mPCR testing should be performed selectively based on patients’ clinical symptoms and diagnoses, and its results should be interpreted with considering identified pathogens. Disclosures All Authors: No reported disclosures

Prevalence of Campylobacter spp. among imported broiler drumsticks and wings sold in Lithuania

The aim of this research was to estimate the prevalence of Campylobacter in imported broiler drumsticks and wings. During the one-year study period, 138 imported broiler samples (raw wings and drumsticks) were collected and tested from 3 different sellers. Campylobacter spp. were detected and isolated using traditional microbiological methods, identified using a multiplex polymerase chain reaction (PCR) method. The results of PCR products were analysed in agarose gel using electrophoresis. After an epidemiological study, C. jejuni and C. coli strains were selected and the prevalence of virulence genes was evaluated. The study identified Campylobacter spp. in 36 (26.1%) samples – 19 raw wings (27.9%) and 17 raw drumsticks (24.3%) samples were infected with these bacteria. Campylobacter spp. were most frequently detected in raw broiler samples during autumn (September–November) (47.2%) and winter (December–February) (41.6%) periods than spring (March–May) (5.5%) or summer (June–August) (5.5%). Contamination of products was not significantly impacted by the sale location (p > 0.05). The examination of virulence factors of Campylobacter spp. revealed that C. jejuni and C. coli strains contain 2 out of 3 virulence genes – CadF and CdtA. The CdtA gene was found in nearly all tested Campylobacter spp. strains isolated from broiler samples (94.4%).

A simple multiplex polymerase chain reaction assay for rapid identification of the common pathogenic dermatophytes:Trichophyton interdigitale, Trichophyton rubrum, and Epidermophyton floccosum

Background and Purpose: The most common etiological agents of human dermatophytosis in various parts of the world are Trichophyton rubrum, Trichophyton interdigitale, and Epidermophyton floccosum. The main aim of this study was to design and evaluate a simple and straightforward multiplex polymerase chain reaction (PCR)assay for reliable identification/differentiation of these species in clinical isolates. Materials and Methods: The reliable sequences of several molecular targets of dermatophytes species were used to design a multiplex PCR for the identification of common pathogenic dermatophytes. The isolates and clinical specimens examined in this study included seven standard strains of dermatophytes, 101 isolates of dermatophytes and non-dermatophyte molds/yeasts which had already been identified by sequencing or PCR-restriction fragment length polymorphism (RFLP), and 155 clinical samples from patients suspected of cutaneous mycoses. Results: Species-specific primer pairs for T. rubrum and T. interdigitale/T. mentagrophytes were designed based on the sequence data of the translation elongation factor 1-alpha gene, and the primers for E. floccosum targeted the specific sequence of the internal transcribed spacer region (ITS). The multiplex PCR successfully detected T.rubrum, T. interdigitale/T. mentagrophytes, and E. floccosum strains that were identified by sequencing or PCR-RFLP. However, the primer pairs selected for T. interdigitale/T. mentagrophytes cross-reacted with Trichophyton tonsurans. In testing the PCR system directly for clinical samples, the proportion of positive multiplex PCR was higher than positive culture (68.1% vs. 55.4%, respectively). Conclusion: The multiplex assay could detect three common agents out of several causal agents of dermatophytosis, namely T. rubrum, T. interdigitale, and E. floccosum.Therefore, by adding pan-dermatophyte primers it can be used as a comprehensive detection/identification test.

An epidemiological survey of Dirofilaria spp. and Acanthocheilonema spp. in dogs from the Republic of Moldova

Background During the last decades, filarial infections caused by Dirofilaria spp. have spread rapidly within dog populations of several European countries. Increasing scientific interest in filariasis, and the availability of new diagnostic tools, has led to improved knowledge of the biology, morphology, and epidemiology of different species of filarial worms. However, data are still scarce for a number of countries, including the Republic of Moldova. Thus, we assessed the epidemiological status of canine filariasis in the Republic of Moldova to address part of this knowledge gap. Methods A total of 120 blood samples were collected between June 2018 and July 2019 from dogs originating from the cities of Cahul and Chişinău. The samples were examined microscopically, and multiplex polymerase chain reaction was performed to evaluate filarioid species diversity. Results Microscopic examination revealed that 12 dogs (10.0%) were positive for circulating microfilariae. The molecular test showed that one dog was positive for Acanthocheilonema reconditum (0.8%), one for Dirofilariaimmitis (0.8%), six for Dirofilariarepens (5.0%), and four (3.3%) harboured a co-infection with D. immitis and D. repens. Prevalence was significantly higher in dogs aged ≥ 2 years. Conclusions The epidemiological survey presented here for the Republic of Moldova confirmed the presence D. immitis, D. repens and A. reconditum in dogs that had not received any heartworm preventive. Graphical abstract