Diagnostic Microbiology & Infectious Disease

Diagnosis of pneumococcal empyema using immunochromatographic test on pleural fluid and serotype distribution in Korean children

Joon-Ho Lee, So Hee Kim, Jina Lee, Eun Hwa Choi, Hoan Jong Lee — 2011-11-14

Abstract: To evaluate the diagnostic value of immunochromatographic test (ICT) on pleural fluid in diagnosing pneumococcal empyema in children and to determine pneumococcal serotypes, 62 exudative parapneumonic effusions from Korean children were tested with culture, ICT for S. pneumoniae, pneumococcal autolysin polymerase chain reaction (PCR), and subsequent sequencing. Of the 62 patients, culture was positive in 3 patients only (4.8%). Pneumococci were identified in 13 samples (21.0%) by sequencing-confirmed PCR and ICT, respectively. When pneumococcal empyema was defined by either positive culture or sequence confirmation, the sensitivity of ICT was 76.9% (10/13) and the specificity of ICT was 93.9%. Eight of 10 patients with positive ICT and culture-negative results had a history of prior antibiotics use, whereas none of the culture-proven cases had. Serotypes of PCR-positive samples were determined by multiplex PCR assays. Multiplex PCR detected serotypes 19A (6), 1 (1), 14 (1), 34 (1), and untypable (4). ICT on pleural fluid is a relatively sensitive and highly specific method for diagnosis of pneumococcal empyema, especially in children given prior antibiotics.

Evaluation of 3 different rapid automated systems for diagnosis of urinary tract infections

2011-11-21

Abstract: Urinary tract infections (UTI) are one of the most common infections in both hospitalised and ambulant patients. Rapid diagnostic of UTIs is necessary to provide early information about the presence of bacteria and the indication to administer an antibiotic therapy. Here we report on a study comparing 3 different rapid automated systems with the semiquantitative plate culture reference method in a university hospital with a highly complex patient population. In total, 2230 urine samples were consecutively tested using the UroQuick (Alifax), the BACSYS-40i (Sysmex), and the UF-1000i (Sysmex) system. In comparison to the results obtained by culture techniques, the automated systems showed a sensitivity of 73.0–80.9% and a specificity ranging between 61.8% and 92.8%. Additionally, sensitivity and specificity for the most common UTI-causing microorganisms were analysed and showed that sensitivity and specificity correlate with the colony forming units of microorganisms in the urine, with a sensitivity of nearly 90% for Gram-negative rods, typical for community acquired UTIs, but a very low sensitivity for Gram-positive bacteria and yeasts. This led us to the conclusion that the currently available automated systems might be rather helpful to analyse a typical UTI in an ambulant patient population but not for rapid diagnosis of UTIs in a complex population of hospitalized patients.

Pyrosequencing-based validation of a simple cell-suspension polymerase chain reaction assay for Campylobacter with application of high-processivity polymerase and novel internal amplification controls for rapid and specific detection

2012-02-01

Abstract: Although Campylobacter is an important food-borne human pathogen, there remains a lack of molecular diagnostic assays that are simple to use, cost-effective, and provide rapid results in research, clinical, or regulatory laboratories. Of the numerous Campylobacter assays that do exist, to our knowledge none has been empirically tested for specificity using high-throughput sequencing. Here we demonstrate the power of next-generation sequencing to determine the specificity of a widely cited Campylobacter-specific polymerase chain reaction (PCR) assay and describe a rapid method for direct cell suspension PCR to quickly and easily screen samples for Campylobacter. We present a specific protocol which eliminates the need for time-consuming and expensive genomic DNA extractions and, using a high-processivity polymerase, demonstrate conclusive screening of samples in <1 h. Pyrosequencing results show the assay to be extremely (>99%) sensitive, and spike-back experiments demonstrated a detection threshold of <102 CFU mL−1. Additionally, we present 2 newly designed broad-range bacterial primer sets targeting the 23S rRNA gene that have wide applicability as internal amplification controls. Empirical testing of putative taxon-specific assays using high-throughput sequencing is an important validation step that is now financially feasible for research, regulatory, or clinical applications.

Low prevalence of Pneumocystis jirovecii lung colonization in Ugandan HIV-infected patients hospitalized with non-Pneumocystis pneumonia

2011-12-07

Abstract: Pneumocystis jirovecii is an important opportunistic infection in human immunodeficiency virus (HIV)–infected patients. In the developed world, P. jirovecii epidemiology is marked by frequent colonization in immunosuppressed patients, but data on the prevalence of colonization are very limited in sub-Saharan Africa, where the majority of persons living with HIV reside. Our objective was to describe the epidemiology of P. jirovecii colonization among HIV-positive patients in a cross-sectional, hospital-based study of patients admitted with suspected pneumonia in Kampala, Uganda. P. jirovecii was detectable in bronchoalveolar lavage fluid from 7 (6%) of 124 consecutive patients with non-Pneumocystis pneumonia. Colonization was not associated with patient demographic or clinical information. This prevalence is substantially lower than in published studies in the developed world and suggests that there is a limited reservoir of organisms for clinical infections in this Ugandan population. These findings may partially explain the low incidence of Pneumocystis pneumonia in Uganda and other sub-Saharan African countries.

Validation of a polymerase chain reaction–oligochromatography test for detection of influenza A (H1N1) 2009 virus

2011-12-05

Abstract: The outbreak of pandemic influenza A (H1N1) 2009 virus caused the first influenza pandemic disease of the 21st century. In August 2010, the pandemic moved into the post-pandemic period. However, localized outbreaks of various magnitudes continued with a higher rate of disease severity. The aim of this study was to assess a new polymerase chain reaction (PCR)–oligochromatographic assay (Speed-Oligo) in the diagnosis of novel influenza A (H1N1) 2009. A total of 405 nasopharyngeal aspirate specimens from 400 pediatric and adults patients with suspected infection of pandemic influenza A (H1N1) 2009 were analyzed. The sensitivity and specificity values of the Speed-Oligo assay in comparison to reverse transcriptase–PCR assay developed by the Centers for Disease Control and Prevention were 86.5% and 92.2%, respectively. The new assay is simple, rapid, and provides a good sensitivity for detection of influenza A (H1N1) 2009. This assay might be a good alternative to real-time PCR assays for laboratories not equipped with real-time PCR instruments.

Evaluation of a novel commercial rapid test for dengue diagnosis based on specific IgA detection

2012-02-01

Abstract: The performance of the novel commercial test ASSURE® Dengue IgA Rapid test (MP Diagnostics) was evaluated using a panel of 172 sera collected from dengue patients and 47 sera from healthy blood donors. The overall specificity and sensitivity were 85.1% and 61.0%, respectively. However, the positivity rate for IgA went from 33.3% for sera collected the same day of fever onset to 81.2% for sera collected 5 days after fever onset. Infections with serotype 2 viruses were detected more efficiently than those with serotype 1 viruses, and no sera from infections with serotypes 3 and 4 were available. In addition, the kit was twice more efficient at detecting secondary infections than at detecting primary infections. Finally, the ASSURE® test showed good repeatability and reproducibility. The results of this study suggest that the ASSURE® Dengue IgA Rapid test may become a useful and easy-to-use test for early dengue diagnosis.

Diagnostic significance of hepatitis B viral antigens in patients with glomerulonephritis-associated hepatitis B virus infection

2012-02-01

Abstract: Hepatitis B viral infection can lead to hepatitis B virus–associated glomerulonephritis, a clinically significant subtype of secondary nephritis. In the present study, we examined the presence of PreS1/S2 antigen in renal tissues by use of immunohistochemistry and investigated the use of PreS1/S2 and 2 HBV serum antigens, HBe-Ag and HBs-Ag, in the diagnosis. We assessed the presence of these 3 antigens in patients with confirmed hepatitis B virus–associated glomerulonephritis (n = 22) and patients without this disease (n = 19). Our results indicate that the combined use of PreS1/S2-Ag and serum HBe-Ag in the diagnosis of hepatitis B virus–associated glomerulonephritis had good positive predictive value (0.89), modest negative predictive value (0.77), and substantial agreement based on Cohen's kappa coefficient (κ = 0.660, P < 0.001). We suggest that our results be considered in the development of more definitive diagnostic criteria for hepatitis B virus–associated glomerulonephritis.

Nontuberculous mycobacterial infections in cancer patients in a medical center in Taiwan, 2005–2008

2011-12-05

Abstract: Data on the nontuberculous mycobacterial (NTM) species that cause infection and the characteristics of disease caused by these pathogens in cancer patients are limited, so we perform this study to investigate the species distribution of NTM isolates from various clinical specimens and to elucidate the epidemiologic trends in NTM isolates and diseases among cancer patients. From 2005 through 2008, cancer patients with NTM infections as defined by the American Thoracic Society/Infectious Diseases Society of America criteria were identified at the National Taiwan University Hospital. The medical records of all patients were reviewed. During the study period, a total of 219 cancer patients with NTM infections were identified. Among them, 133 (60.7%) patients were older than 65 years, most of whom were men. Lung cancer was the most common type of cancer, followed by hematologic cancer and gastrointestinal tract cancer. Pulmonary NTM infection was the most common type of infection in 205 (93.6%) patients, followed by skin and soft tissue infections (n = 7, 3.2%), disseminated infections (n = 4, 1.8%), and genitourinary tract infection (n = 3, 1.4%). Disseminated infections occurred exclusively in patients with hematologic cancer. Mycobacterium avium complex (MAC) caused the majority of pulmonary NTM infections in cancer patients; in contrast, M. abscessus was the most common causative pathogen of extrapulmonary NTM diseases, followed by MAC. In conclusion, physicians need to be aware of the possibility of co-existing pulmonary NTM infection in patients with lung cancer. In addition, disseminated NTM infection should be considered in patients with hematologic cancer.

Development of an ultrasensitive polymerase chain reaction–amplified immunoassay based on mycobacterial RD antigens: implications for the serodiagnosis of tuberculosis

Promod K. Mehta, Mamta Kalra, Gopal Krishan Khuller, Digambar Behera, Indu Verma — 2012-02-01

Abstract: Immuno-polymerase chain reaction (I-PCR) combines the versatility of enzyme-linked immunosorbent assay (ELISA) with the exponential amplification power of PCR. The present study was designed to detect antibodies to Mycobacterium tuberculosis complex–specific region of difference (RD) antigens, i.e., early secretory antigenic target-6, culture filtrate protein-10, culture filtrate protein-21, and mycobacterial protein from species tuberculosis-64, as well as antigens in pulmonary tuberculosis patients by I-PCR assay. We could detect ESAT-6 and other RD antigens up to 0.1 fg by I-PCR assay, thus resulting in 107 times higher sensitivity than that observed with ELISA. With paired sample analysis based on the detection of antibodies in serum and antigens in sputum of the same individual, the sensitivity of RD multi-antigen cocktail-based I-PCR assay was 72% in smear-negative cases and 91% in smear-positive cases of pulmonary tuberculosis with high specificity values. In extrapulmonary tuberculosis patients, higher sensitivity was observed by detecting cocktail of antigens by I-PCR assay as compared to sensitivity earlier observed in the same samples by ELISA.

Molecular diagnosis and species identification of imported malaria in returning travellers in Italy

2011-11-14

Abstract: A new seminested polymerase chain reaction (sn-PCR)–based protocol was developed and used to detect and identify Plasmodium species in 1226 whole-blood samples from patients (872 Italians and 354 foreigners) with at least 1 symptom compatible with clinical malaria. The results were compared with those obtained by microscopy: 187 samples were positive by microscopy for malaria parasites and 196 were positive by sn-PCR. When compared to microscopy, the sn-PCR detected different malaria parasite species in 11 cases. In 4 of 11 cases, the sn-PCR identified 1 additional malaria parasite species not observed microscopically, suggesting increased sensitivity. In 4 samples with levels of parasitemia too low for accurate identification of species by microscopy, the sn-PCR detected 2 P. falciparum, 1 P. ovale, and 1 P. falciparum plus P. ovale. Moreover, 9 negative samples by microscopy were positive by sn-PCR. Follow-up analysis demonstrated a parasite clearance of P. falciparum DNA up to 3 days after the disappearance of parasitemia at microscopy. In conclusion, sn-PCR–based diagnosis of malaria appears to be a useful tool when the results of conventional techniques are negative in the presence of a syndrome consistent with malaria, yielding accurate species identification and consequential correct treatment.

Fulminant gestational hepatitis due to primary herpes simplex type 2 infection: use of serum HSV polymerase chain reaction for noninvasive diagnosis

2011-11-21

Abstract: Acute gestational hepatitis from herpes simplex virus (HSV) infection is a rare but potentially life-threatening condition. We present the first reported case of primary HSV type 2 hepatitis in a pregnant woman who was diagnosed by detection of HSV-2 viremia via real-time polymerase chain reaction. The patient was successfully treated with acyclovir and delivered a healthy infant.

Coccidioidomycosis of cervical lymph nodes in an HIV-infected patient with immunologic reconstitution on potent HAART: a rare observation in a nonendemic area

2011-11-21

Abstract: Coccidioidomycosis is caused by the dimorphic fungus Coccidioides immitis, which is endemic in southwestern United States. We report a case of coccidioidomycosis of cervical lymph nodes that occurred early after the introduction of highly active antiretroviral therapy during the phase of immune system recovery, demonstrating a rare disease in a nonendemic area.

Clinical evidence for rapid transmission of Lyme disease following a tickbite

Eleanor D. Hynote, Phyllis C. Mervine, Raphael B. Stricker — 2011-11-21

Abstract: Lyme disease transmission to humans by Ixodes ticks is thought to require at least 36–48 h of tick attachment. We describe 3 cases in which transmission of Borrelia burgdorferi, the spirochetal agent of Lyme disease, appears to have occurred in less than 24 h based on the degree of tick engorgement, clinical signs of acute infection, and immunologic evidence of acute Lyme disease. Health care providers and individuals exposed to ticks should be aware that transmission of Lyme disease may occur more rapidly than animal models suggest. A diagnosis of Lyme disease should not be ruled out based on a short tick attachment time in a subject with clinical evidence of B. burgdorferi infection.

Methicillin-resistant Staphylococcus aureus expressing low-level methicillin resistance may not be detected by the VITEK2® system

2011-11-21

Abstract: Low-level methicillin-resistant Staphylococcus aureus may be difficult to detect with the VITEK® 2 system (VK2). Here, we suggest that S. aureus exhibiting VK2-oxacillin MIC of 1 or 2 mg/L and a negative cefoxitin screen should be tested for the presence of mecA or its gene product.

Detection of 10 medically important Candida species by seminested polymerase chain reaction

Leslie Thian Lung Than, Pei Pei Chong, Kee Peng Ng, Heng Fong Seow — 2011-12-12

Abstract: A seminested PCR detecting ten medically important Candida species were achieved. Analytical sensitivity and specificity were not compromised.