Sensitivity and specificity of the Leishmania OligoC‐TesT and NASBA‐oligochromatography for diagnosis of visceral leishmaniasis in Kenya

Objective To estimate the sensitivity and specificity of the OligoC‐TesT and nucleic acid sequence‐based amplification coupled to oligochromatography (NASBA‐OC) for molecular detection of Leishmania in blood from patients with confirmed visceral leishmaniasis (VL) and healthy endemic controls from Kenya. Methods Blood specimens of 84 patients with confirmed VL and 98 endemic healthy controls from Baringo district in Kenya were submitted to both assays. Results The Leishmania OligoC‐TesT showed a sensitivity of 96.4% (95% confidence interval [CI]: 90–98.8%) and a specificity of 88.8% (95% CI: 81–93.6%), while the sensitivity and specificity of the NASBA‐OC were 79.8% (95% CI: 67–87%) and 100% (95% CI: 96.3–100%), respectively. Conclusion Our findings indicate high sensitivity of the Leishmania OligoC‐TesT on blood while the NASBA‐OC is a better marker for active disease.     

Using the full spectral capacity (six channels) of a real-time PCR instrument can simplify diagnostic laboratory screening and typing protocols for pandemic H1N1 influenza

Please cite this paper as: Hopkins et al. (2011) Using the full spectral capacity (six channels) of a real‐time PCR instrument can simplify diagnostic laboratory screening and typing protocols for pandemic H1N1 influenza. Influenza and Other Respiratory Viruses 5(2), 110–114. Background Timely reporting of influenza A virus subtype affects patient management. Real‐time PCR is a rapid and sensitive method routinely used to characterise viral nucleic acid, but the full spectral capability of the instruments is not employed. Objectives To evaluate a hexaplex real‐time PCR assay (Flu‐6plx assay) capable of detecting influenza A and B, hMPV, respiratory syncytial virus (RSV) and distinguishing 2008 ‘human’ influenza A/H1 from 2009 pandemic A/H1 subtypes. Methods Respiratory specimens (n = 213) were tested using the Flu‐6plx assay and a further four monoplex PCRs targeting hMPV, RSV, influenza A and B. The FDA‐approved ProFlu ST test was used to validate the Flu‐6plx PCR influenza A/H1 subtyping components. Discrepant 2009 pandemic A/H1 results were further tested using the CDC swine H1 assay. Results The Flu‐6plx assay had excellent sensitivity identifying 106/106 influenza A RNA–positive samples. The ProFlu ST test was a less sensitive subtyping test, and discrepant analysis could not confirm A/H1 status for four samples resulting in Flu‐6plx PCR specificities of 98% and 95% for human A/H1 and 2009 pandemic A/H1, respectively. Co‐infection affected the sensitivity of the Flu‐6plx PCR hMPV component whereby low‐level hMPV RNA could be masked by much higher concentrations of influenza A virus RNA. Conclusions The Flu‐6plx assay is a sensitive and specific test for the universal detection of influenza A infection and determination of A/H1 subtype. Concomitant detection of influenza B, hMPV and RSV demonstrates the utility of hexaplex real‐time PCRs in viral diagnostics.     

Comparison of a singleplex real-time RT-PCR assay and multiplex respiratory viral panel assay for detection of influenza "A" in respiratory specimens

Please cite this paper as: Pabbaraju et al. (2011) Comparison of a singleplex real‐time RT‐PCR assay and multiplex respiratory viral panel assay for detection of influenza “A” in respiratory specimens. Influenza and Other Respiratory Viruses 5(2), 99–103. Background Evaluation of different molecular tests for the detection of pandemic (H1N1) 2009 virus is important before the next wave of the pandemic. Objectives To compare a hydrolysis probe‐based real‐time RT‐PCR assay recommended by the CDC to the xTAG^® respiratory viral panel (RVP) (Luminex^® Molecular Diagnostics) for the detection of influenza A. Methods Eleven thousand eight hundred and ninety‐eight respiratory specimens were tested by the real‐time RT‐PCR and RVP assays for the detection of influenza A. The distribution of seasonal H1, H3 and pandemic H1N1 subtypes in these specimens was compared. Results The RVP assay was generally unable to identify influenza A–positive samples with a low viral load, whereas the real‐time RT‐PCR assay detected most of these samples resulting in a subset of specimens that could not be confirmed as either seasonal or pandemic influenza A subtypes. Conclusions When the prevalence of influenza A is high, the CDC recommended real‐time RT‐PCR has significant advantages as a frontline assay, namely higher sensitivity and shorter time to reporting a result. Anticipated scenarios would be during the peaks of the pandemic and episodes of seasonal influenza. Furthermore, the better sensitivity of the RT‐PCR makes it the preferred assay to detect influenza in patients with severe respiratory disease tested late in their clinical course. If pandemic (H1N1) 2009 virus is not the dominant virus and there is a high proportion of other respiratory viruses circulating, laboratories will be faced with the decision to use the RVP assay for the detection of pandemic (H1N1) 2009 virus.     

Schistosoma real‐time PCR as diagnostic tool for international travellers and migrants

Objective To evaluate the use of a genus‐specific PCR that combines high sensitivity with the detection of different Schistosoma species for diagnosis in international travellers and migrants in comparison to standard microscopy. Methods and results The genus‐specific real‐time PCR was developed to target the 28S ribosomal RNA gene of the major human Schistosoma species. It was validated for analytical specificity and reproducibility and demonstrated an analytical sensitivity of 0.2 eggs per gram of faeces. Its diagnostic performance was further evaluated on 152 faecal, 32 urine and 38 serum samples from patients presenting at the outpatient clinic of the Institute of Tropical Medicine in Antwerp (Belgium). We detected Schistosoma DNA in 76 faecal (50.0%) and five urine (15.6%) samples of which, respectively, nine and one were not detected by standard microscopy. Only two of the 38 serum samples of patients with confirmed schistosomiasis were positive with the presently developed PCR. Sequence analysis on positive faecal samples allowed identification of the Schistosoma species complex. Conclusion The real‐time PCR is highly sensitive and may offer added value in diagnosing imported schistosomiasis. The genus‐specific PCR can detect all schistosome species that are infectious to humans and performs very well with faeces and urine, but not in serum.     

Reproducibility of multiple breath washout indices in the unsedated preterm neonate

Multiple breath inert gas washout (MBW) is gaining popularity for measurements of resting lung volume and ventilation inhomogeneity. Test reproducibility is an important determinant of the clinical applicability of diagnostic tests. The between‐test reproducibility of variables derived from MBW tests in newborn infants is unknown. We aimed to determine the within‐test repeatability and short‐term between‐test reproducibility of MBW variables in unsedated preterm infants. We hypothesized that measurements obtained within a 3‐day interval in clinically stable preterm infants would be reproducible and suitable for use as an objective clinical outcome measurement. In this cross‐sectional observational study, clinically stable hospitalized preterm infants whose parents had given informed consent for MBW studies were tested twice within 72 hr during quiet, unsedated sleep. Functional residual capacity (FRC), lung clearance index (LCI), and the first and second to zeroeth moment ratios (M₁:M₀; M₂:M₀) were computed from MBW traces obtained using a mainstream ultrasonic flowmeter and 4% sulphur hexafluoride (MBW_SF6). Within‐test repeatability and between‐test reproducibility were determined. Within‐test repeatability (expressed as a coefficient of variability (C_v)) for differences between two and four replicate measurements on the same test occasion, were 9.3% (FRC), 9.0% (LCI), 7.6% (M₁:M₀), and 15.6% (M₂:M₀), respectively. The within‐test C_v's were not statistically different to the between‐tests C_v's, which were 7.7% (FRC), 10.3% (LCI), 6.1% (M₁:M₀), and 13.0% (M₂:M₀), respectively. Among unsedated preterm infants, between‐test reproducibility over a 3‐day interval was similar to within‐test repeatability. The wide limits of agreement may limit the application of these measures to detect a clinically significant change in condition in small preterm infants. Pediatr Pulmonol. 2010; 45:62–70. © 2009 Wiley‐Liss, Inc.     

Serological profiles and evaluation of parasitaemia by PCR and blood culture in individuals chronically infected by Trypanosoma cruzi treated with benzonidazole

Objective To evaluate the serological and parasitological status of patients with chronic Chagas disease (CD) after chemotherapy with benzonidazole. Methods Retrospective study of patients treated with benzonidazole (5 mg/kg/day for 60 days) between 1980 and 2010. Twenty‐nine patients who had CD confirmed by two reagent immunological tests and/or one positive xenodiagnosis before treatment were included. Conventional serology (ELISA and IIF) and parasitological tests (haemoculture and N‐PCR) were performed. Results At the time of treatment, the mean age of patients was 36 ± 7.24 years (20–39 years) and the time post‐treatment varied from 1 to 29 years. After chemotherapy, all individuals had reagent ELISA and 93.1% had positive results for the IIF test. T. cruzi DNA was detected by N‐PCR in 48.3%. Negative results were observed in 41.4% and inconclusive ones in 10.3%. Haemoculture was negative for all individuals. Conclusions Our results suggest that N‐PCR may be useful in the early identification of therapeutic failure of CD. Although it is difficult to determine parasitological cure in negative N‐PCR cases, we can infer that this condition represents a declination of parasitaemia as a favourable consequence of aetiological treatment.     

Influence of infection on malaria‐specific antibody dynamics in a cohort exposed to intense malaria transmission in northern Uganda

The role of submicroscopic infections in modulating malaria antibody responses is poorly understood and requires longitudinal studies. A cohort of 249 children ≤5 years of age, 126 children between 6 and 10 years and 134 adults ≥20 years was recruited in an area of intense malaria transmission in Apac, Uganda and treated with artemether/lumefantrine at enrolment. Parasite carriage was determined at enrolment and after 6 and 16 weeks using microscopy and PCR. Antibody prevalence and titres to circumsporozoite protein, apical membrane antigen‐1 (AMA‐1), merozoite surface protein‐1 (MSP‐1₁₉), merozoite surface protein‐2 (MSP‐2) and Anopheles gambiae salivary gland protein 6 (gSG6) were determined by ELISA. Plasmodium falciparum infections were detected in 38·1% (194/509) of the individuals by microscopy and in 57·1% (284/493) of the individuals by PCR at enrolment. Antibody prevalence and titre against AMA‐1, MSP‐1₁₉, MSP‐2 and gSG6 were related to concurrent (sub‐)microscopic parasitaemia. Responses were stable in children who were continuously infected with malaria parasites but declined in children who were never parasitaemic during the study or were not re‐infected after treatment. These findings indicate that continued malaria infections are required to maintain antibody titres in an area of intense malaria transmission.     

Detection of mutant P2 adenosine transporter (TbAT1) gene in Trypanosoma brucei gambiense isolates from northwest Uganda using allele-specific polymerase chain reaction

Objective To assess the application of allele‐specific PCR (AS‐PCR) as a fast, cheap and reliable method for detecting mutant TbAT1 associated with melarsoprol relapse in Trypanosoma brucei gambiense isolates from northwest Uganda. Methods A total of 105 trypanosome isolates were analysed using SfaN1 restriction fragment length polymorphism (RFLP) and AS‐PCR, the former used as the gold standard. Sensitivity, specificity, positive and negative predictive values of AS‐PCR as well as agreement between the tests were determined. Results Eleven trypanosome isolates had mutant TbAT1 while 94 exhibited the wild‐type TbAT1 genes. There was a highly significant agreement between SfaN1 RFLP and AS‐PCR with kappa and intra‐class correlation values of 1.0. The sensitivity and specificity of AS‐PCR were both 100%, while the positive and negative predictive values were found to be equal to 1.0. Cost and time analyses were performed and AS‐PCR was 4.3 times cheaper than SfaN1 RFLP, in addition to the less time required for its execution. Conclusion AS‐PCR should be the test of choice for screening for mutant TbAT1 in the ever‐increasing numbers of field trypanosome isolates.     

Evaluation of two rK39 dipstick tests, direct agglutination test, and indirect fluorescent antibody test for diagnosis of visceral leishmaniasis in a new epidemic site in highland Ethiopia

We assessed the performance characteristics of two rK39 immunochromatographic tests, a direct agglutination test (DAT), and an indirect immunofluorescent antibody test (IFAT) in the site of a new epidemic of visceral leishmaniasis (VL) in northwestern Ethiopia. The study population was composed of 179 patients with suspected VL and 67 controls. The sensitivities of Kalazar Detect^®, DiaMed-IT Leish^®, DAT, and IFAT in 35 polymerase chain reaction–confirmed VL cases were 94.3%, 91.4%, 91.4%, and 100%, respectively, and the specificities were 98.5%, 94%, 98.5%, and 98.5%, respectively. In a Bayesian latent class analysis of all 246 specimens, the estimated sensitivities were 90.5%, 89%, 88.8%, and 96% for Kalazar Detect^®, DiaMed-IT Leish^®, DAT, and IFAT, respectively; DAT showed the highest estimated specificity (97.4%). Both rK39 immunochromatographic tests perform as well as DAT, and are suitable for VL diagnosis in first-level health centers in this area of Ethiopia.     

Establishment and evaluation of a 30-minute detection method for SARS-CoV-2 nucleic acid using a novel ultra-fast real-time PCR instrument

BackgroundThe coronavirus disease 2019 (COVID-19) pandemic is still raging worldwide. Efficient, fast and low-cost severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid detection methods are urgently needed.MethodsA rapid PCR temperature change mode was explored by moving the reaction tube between the independent temperature modules with large temperature differences and a portable ultra-fast real-time PCR instrument were developed. We established a rapid SARS-CoV-2 test method using the ultra-fast real-time PCR instrument, a China Food and Drug Administration-certified SARS-CoV-2 reagent and optimized reaction condition. The analytical and clinical performances of the rapid tests were evaluated by comparing with the standard SARS-CoV-2 tests.ResultsThe new temperature change mode can effectively shorten the amplification reaction time and be successfully used in the development of the ultra-fast real-time PCR instrument. The rapid SARS-CoV-2 test method was established and the time to yield results were greatly shortened from 81 min of the standard test to 31 min. Specificity of the rapid test was assessed and no non-specific amplification (0/63) was observed. The limits of detection of the rapid and standard tests were similar. Clinical performance was evaluated using 184 respiratory specimens from patients with suspected SARS-CoV-2 infection. The positive agreement between the rapid and standard tests was 100% (67/67), the negative agreement was 97.4% (114/117), and the kappa statistic was 0.965 (P<0.001). No significant differences in the Ct values for each target gene were observed between the rapid test and the standard test (P>0.05).ConclusionsWe had developed a 30-minute detection method for SARS-CoV-2 nucleic acid using a novel ultra-fast real-time PCR instrument. The rapid test method may impact on patient management.     

The pp67 mRNA assay in treatment and monitoring of cytomegalovirus disease in renal transplant patients in India

The present report describes use of nucleic acid sequence-based amplification (NASBA) technology to detect pp67 mRNA of cytomegalovirus (CMV) in transplant patients in India. In our experience, pp67 mRNA assay was an accurate, rapid, and effective diagnostic tool to detect active CMV disease in 40.7% (50/123) of symptomatic transplant cases. This assay also allowed us to monitor CMV therapy. As part of the immunosuppressive regimen mycophenolate mofetil was found to increase the risk of developing CMV disease. All positive cases with this assay were subjected to antiviral therapy, with complete remission of the disease. At our center CMV NASBA assay has become the gold standard for the diagnosis of CMV disease in transplant patients.     

Evaluating the risk of hepatitis B reactivation in patients with haematological malignancies: is the serum hepatitis B virus profile reliable?

Background/Aim: Patients with an occult hepatitis B virus (HBV) infection undergoing deep immunosuppression are potentially at risk of HBV reactivation. In order to assess whether a polymerase chain reaction (PCR) assay for HBV DNA in serum could be used to predict the reactivation of an occult HBV infection, we performed a retrospective study in a cohort of Sicilian patients with oncohaematological diseases. Methods: We studied by a highly sensitive ad hoc nested PCR for serum HBV DNA 75 HBsAg‐negative oncohaematological patients requiring chemotherapy. Results: Thirty‐three patients (44%) were HBV seronegative (anti‐HBc and anti‐HBs negative) and 42 patients (56%) were HBV seropositive (anti‐HBc and/or anti‐HBs positive). Baseline serum HBV DNA was positive in nine out of 33 HBV‐seronegative patients and in nine out of 42 HBV‐seropositive patients (27.3 vs. 21.4%; P=NS). HBsAg seroconversion was observed in five out of 33 seronegative vs. six out of 42 seropositive patients (15 vs. 14%, P=0.9), and in five out of 18 HBV DNA‐positive vs. six out of 57 HBV DNA‐negative patients (27.7 vs. 10.6%P=0.11). Hepatitis C virus infection was found in 18 patients (24.3%), although with no correlation to HBV serological status, presence of serum HBV DNA or frequency of HBsAg seroconversion. Conclusions: In oncohaematological patients undergoing chemotherapy, highly sensitive serum HBV DNA testing at baseline has a 28% predictive ability to forecast HBsAg seroconversion in HBV DNA‐positive patients, and a 90% ability to forecast persistent HBsAg negativity in HBV DNA‐negative patients, a better performance than serological tests.     

Detection of drug-resistant HIV minorities in clinical specimens and therapy failure

Objective

Particularly for therapy‐experienced patients, resistance assessment by genotypic or phenotypic methods produces discordances. This study seeks proof that differences may arise from the fact that genotyping produces a single summary sequence whereas replicative phenotyping (rPhenotyping) functionally detects and assigns resistances in mixed HIV populations.

Methods

For validation, defined mixes of wild‐type and M184V mutant were analysed by rPhenotyping or standard genotyping. Allele‐specific and quantitative polymerase chain reaction (PCR) set detection and quantification limits for minor virus populations in vitro and in authentic clinical samples showing geno‐/pheno‐discrepant lamivudine resistance.

Results

Allele‐specific and real‐time PCR methods detected down to 0.3% of mutant M184V. The functional assessment was sensitive enough to reveal <1% of mutant M184V in mixed samples. Also in discordant samples from the diagnostic routine, in which rPhenotyping had identified drug resistance, real‐time PCR confirmed minute amounts of mutant M184V.

Conclusion

By utilizing the replication dynamics of HIV under drug pressure, a rPhenotyping format potently reveals relevant therapy‐resistant minority species, even of HIV known to possess reduced replicative fitness. With its rapid turnaround of 8 days and its high sensitivity, our rPhenotyping system may be a valuable diagnostic tool for detecting the early emergence of therapy‐threatening HIV minorities or the persistence of residual resistant virus.     

实时荧光定量PCR在手足口病肠道病毒快速检测中的应用

目的建立快速检测手足口病肠道病毒的实时荧光定量PCR法,并作出应用分析。方法采用卫生部《手足口病预防控制指南》(2008年版)推荐的RT-PCR法,对丽水市2008年4～5月间发生的172例临床诊断手足口病患者的324份标本进行人肠道病毒、柯萨奇病毒A组16型和肠道病毒EV71型特异性核酸的检测,同时采用实时荧光定量PCR法进行平行检测,比较两者的实验结果,分析实时荧光定量PCR方法的特异性和灵敏度。结果实时荧光定量PCR检测324份标本,肠道病毒通用核酸阳性70份,柯萨奇A16核酸阳性22份,肠道EV71病毒核酸阳性15份,与RT-PCR结果一致,符合率100%。结论实时荧光定量PCR法具有特异性强、灵敏度高等优点,是一种理想的手足口病肠道病毒的检测方法。     

Detection of clarithromycin resistance in Helicobacter pylori following noncryogenic storage of rapid urease tests for 30 days

OBJECTIVE: Traditional Helicobacter pylori (H. pylori) eradication therapy has been undermined by increasing antimicrobial, especially clarithromycin, resistance. Susceptibility testing in some areas is difficult to achieve or unavailable. We aimed to determine whether gastric biopsy specimens stored at room temperature for rapid urease test (RUT) were suitable for clarithromycin susceptibility testing of H. pylori. METHODS: After 30 days of storage at room temperature, DNA was extracted from gastric biopsies present in RUTs (Hpfast). H. pylori status and clarithromycin susceptibility were evaluated using H. pylori‐specific polymerase chain reaction (PCR) for ureA, vacA, and allele‐specific primer‐PCR of the 23S rRNA genes. The PCR results were compared with histology, RUT, and culture results. H. pylori positive was defined as RUT and either culture or histology positive; H. pylori negative as RUT, culture and histology negative. RESULTS: Samples from 31 patients were evaluated; 11 were H. pylori positive including 9 by culture; seven of which had allele‐specific primer‐PCR results from the RUT specimen for the detection of mutations of the 23S rRNA gene. When both tests were available, culture and PCR results were concordant in 7 cases. In 15 of the 20 histology, RUT and culture negative patients, three PCR were negative. In one patient, all of the three tests were positive; and in three only the 23S rRNA was positive and in one only ureA was positive. CONCLUSION: Gastric biopsy specimens stored in the gel of RUT for 30 days can be used for molecular testing to confirm the diagnosis of H. pylori infection and test for clarithromycin susceptibility.     

Identification of Old World Leishmania species by PCR–RFLP of the 7 spliced leader RNA gene and reverse dot blot assay

The aim of the study was to assess the 7SL RNA PCR followed by restriction fragment length polymorphism (RFLP) and reverse dot blot (RDB) assays for use in identification of Old World Leishmania species. Species‐specific RFLP patterns were obtained for Leishmania major, Leishmania tropica and the Leishmania donovani complex when the 7SL RNA PCR product was digested with the restriction enzyme BsuRI, an isoschizomer of HaeIII. For the RDB assay, biotin‐labelled 7SL RNA amplicons were hybridized to Leishmania genus‐specific and species‐specific oligonucleotide probes immobilized onto a membrane. The Old World Leishmania species could be distinguished by using five probes: one that was a genus‐specific probe and hybridized to all Leishmania species (Lc), two that were specific for L. major (Lm1 and Lm2), one that was specific for L. tropica (Lt) and one that detected both L. major and L. tropica (Lmt). The PCR–RDB was 10 times more sensitive than 7SL PCR and can detect <1 parasite. In addition, the identification of species was easier and more reliable than with 7SL PCR–RFLP. 7SL PCR–RFLP detected parasites in 50 of 57 clinical samples, whereas PCR–RDB detected 53 and 55 were detected by amplification of kinetoplast (k) DNA. The 7SL RNA PCR has proven useful for direct diagnosis of Old World leishmaniasis, especially when combined with the RBD assay for species identification.