Serum interleukin-6, procalcitonin, and C-reactive protein at hospital admission can identify patients at low risk for severe COVID-19 progression.

Background: COVID-19 can show a variable course, from asymptomatic infections to acute respiratory failure and death. For efficient allocation of resources, patients should be stratified according to their risk for a severe course as early as possible. Methods: 135 hospitalized patients with COVID-19 pneumonia at four German hospitals were prospectively included in this observational study. A standardized clinical laboratory profile was taken at hospital admission and a panel of serum markers with possible roles in the COVID-associated cytokine storm were also determined. 112 patients could be evaluated. The primary endpoint of ventilator requirement or death within 30 days of symptom onset was met by 13 patients. Results: Serum elevations of interleukin-6 (IL-6), procalcitonin (PCT), and C-reactive protein (CRP) at hospital admission were each highly significantly (p < 0.001) associated with ventilator requirement/death within 30 days of symptom onset. With a sensitivity of 92% and a specificity of 65-67%, IL-6 ≥ 52.8 pg/ml, PCT ≥ 0.11 ng/ml, and CRP ≥ 71.1 mg/L were predictive of a severe course of COVID-19. Positive likelihood ratios were between 2.6-2.8 and negative likelihood ratios were between 0.11-0.13 for these three markers. Conclusion: Negative likelihood ratios indicate that IL-6, PCT, and CRP at hospital admission can be used for identifying patients at low risk for severe COVID-19 progression.

Very low prevalence of Mansonella perstans-specific cell-free DNA in serum samples of Ghanaian HIV patients.

Background: Mansonellosis is a widely neglected helminth disease which is predominantly observed in tropical regions. This study was conducted to assess potential associations of the prevalence of circulating Mansonella perstans-specific cell-free DNA in human serum and HIV infection in Ghanaian individuals. Methods: For this purpose, serum samples obtained from Ghanaian HIV-patients (n = 989) and non-HIV-infected Ghanaian control individuals (n = 91) were subjected to real-time PCR targeting the ITS-(internal transcribed spacer-)2 sequence of M. perstans and Mansonella sp. Deux. Results: Mansonella-specific cell-free DNA was detected in serum samples of only 2 HIV-positive and 0 HIV-negative individuals, making any reliable conclusions on potential associations between HIV and mansonellosis in tropical Ghana unfeasible. Conclusions: Future epidemiological studies on hypothetical associations between mansonellosis and HIV infections should focus more specifically on high-endemicity settings for both Mansonella spp.-infections and HIV-infections, include higher case numbers and be based on real-time PCR from whole blood rather than from serum, in which only circulating parasite DNA but no more cell-bound parasite DNA can be detected. However, the study did not show associations of HIV infections in Ghanaian individuals with Mansonella worm loads high enough to detect cell-free Mansonella DNA in serum by PCR.

Comparative Assessment of Two Commercial Real-Time PCR Assays for the Diagnosis of Trypanosoma cruzi DNA in Serum.

This study was performed to comparably assess two commercial real-time PCR assays for the identification of Trypanosoma cruzi DNA in serum. A total of 518 Colombian serum samples with high pre-test probability for infections with either T. cruzi or apathogenic Trypanosoma rangeli were assessed. The assessment comprised the NDO real-time PCR (TIB MOLBIOL, ref. no. 53-0755-96, referred to as the TibMolBiol assay in the following) with specificity for T. cruzi and the RealStar Chagas PCR Kit 1.0 (altona DIAGNOSTICS, order no. 611013, referred to as the RealStar assay in the following) targeting a kinetoplast sequence of both T. cruzi and T. rangeli without further discrimination. To discriminate between T. cruzi- and T. rangeli-specific real-time PCR amplicons, Sanger sequencing results were available for a minority of cases with discordant real-time PCR results, while the amplicons of the remaining discordant samples were subjected to nanopore sequencing. The study assessment indicated a proportion of 18.1% (n = 94) T. cruzi-positive samples next to 24 samples (4.6%) containing DNA of the phylogenetically related but apathogenic parasite T. rangeli. The observed diagnostic accuracy as expressed by sensitivity and specificity was 97.9% (92/94) and 99.3% (421/424) with the TibMolBiol assay and 96.8% (91/94) and 95.0% (403/424) with the RealStar assay, respectively. Reduced specificity resulted from cross-reaction with T. rangeli in all instances (3 cross-reactions with the TibMolBiol assay and 21 cross-reactions with the RealStar assay). DNA from the six discrete typing units (DTUs) of T. cruzi was successfully amplified by both real-time PCR assays. In summary, both assays showed a comparable diagnostic accuracy for the diagnosis of T. cruzi from human serum, with a slightly higher specificity seen for the TibMolBiol assay. The pronounced co-amplification of DNA from apathogenic T. rangeli according to the RealStar assay may be a disadvantage in areas of co-circulation with T. cruzi, while the test performance of the two compared assays will be quite similar in geographic settings where T. rangeli infections are unlikely.

Intestinal Helminth Infections in Ghanaian Children from the Ashanti Region between 2007 and 2008-A Retrospective Cross-Sectional Real-Time PCR-Based Assessment.

In spite of ongoing eradication programs, helminth infections are still a medical issue in Ghana. For follow-up assessments on the decline of regional helminth infections, historic baseline prevalence values obtained with standardized diagnostic procedures can be helpful. In this retrospective cross-sectional study, real-time PCR targeting the nematodes Ancylostoma spp. (ITS2), Ascaris lumbricoides (ITS1), Enterobius vermicularis (ITS1), Necator americanus (ITS2), Strongyloides stercoralis (18S rRNA) and Trichuris trichiura (18S rRNA), the trematodes Schistosoma spp. (ITS2) as well as the cestodes Hymenolepis nana (ITS1), Taenia saginata (ITS1) and Taenia solium (ITS1) was applied with 2046 DNA eluates from stool samples of Ghanaian children from the Ashanti region collected between 2007 and 2008 in order to retrospectively define prevalence values. The overall prevalence was low with 3.8% (n = 77) and only 0.1% (n = 2) double infections with helminths were recorded. The three most frequently detected enteric helminth species comprised 2% S. stercoralis (n = 41), 0.8% H. nana (n = 16), and 0.7% N. americanus (n = 14), while only sporadic infection events were recorded for other helminth species comprising 0.1% E. vermicularis (n = 2), 0.1% Schistosoma spp. (n = 2), 0.1% T. saginata (n = 1) and 0.1% T. trichiura (n = 1). A. lumbricoides, Ancylostoma spp. and T. solium were not detected at all. In conclusion, the retrospective assessment suggests a low prevalence of enteric helminth infections in Ghanaian children from the Ashanti Region within the assessment period between 2007 and 2008.

Metagenomic Sequencing for the Diagnosis of Plasmodium spp. with Different Levels of Parasitemia in EDTA Blood of Malaria Patients-A Proof-of-Principle Assessment.

Molecular diagnostic approaches are increasingly included in the diagnostic workup and even in the primary diagnosis of malaria in non-endemic settings, where it is difficult to maintain skillful microscopic malaria detection due to the rarity of the disease. Pathogen-specific nucleic acid amplification, however, bears the risk of overlooking other pathogens associated with febrile illness in returnees from the tropics. Here, we assessed the discriminatory potential of metagenomic sequencing for the identification of different Plasmodium species with various parasitemia in EDTA blood of malaria patients. Overall, the proportion of Plasmodium spp.-specific sequence reads in the assessed samples showed a robust positive correlation with parasitemia (Spearman r = 0.7307, p = 0.0001) and a robust negative correlation with cycle threshold (Ct) values of genus-specific real-time PCR (Spearman r = -0.8626, p ≤ 0.0001). Depending on the applied bioinformatic algorithm, discrimination on species level was successful in 50% (11/22) to 63.6% (14/22) instances. Limiting factors for the discrimination on species level were very low parasitemia, species-depending lacking availability of reliable reference genomes, and mixed infections with high variance of the proportion of the infecting species. In summary, metagenomic sequencing as performed in this study is suitable for the detection of malaria in human blood samples, but the diagnostic detection limit for a reliable discrimination on species level remains higher than for competing diagnostic approaches like microscopy and PCR.

Comparison of the Influence of Different Nucleic Acid Extraction Assays on the Sensitivity of Trypanosoma cruzi-Specific Real-Time PCR.

For the molecular diagnosis of Chagas disease by real-time PCR (polymerase chain reaction), optimization of diagnostic accuracy is desirable. The detection limit of real-time PCR assays for the diagnosis of Trypanosoma cruzi in human serum is affected by various influences including the choice of the nucleic acid extraction assay. In this study, three nucleic acid extraction assays were compared regarding their influence on the sensitivity of a T. cruzi-specific real-time PCR with 62 reference sera containing T. cruzi target DNA (deoxyribonucleotide acid). More than 95% of the positive sera were correctly identified after all three nucleic acid extraction strategies with a detection rate ranging from 96.8% (60/62) for the worst assay to 100% (62/62) for the best one. A matched pairs analysis for the comparison of the cycle threshold (Ct) values obtained with the 59 reference samples with positive real-time PCR results after all three nucleic acid extraction schemes indicated differences in a range of about 3 Ct steps. Summarized, all three compared nucleic acid extraction schemes were basically suitable for T. cruzi-specific PCR from serum with some minor differences. However, in the case of low quantities of circulating parasite DNA in the serum of a patient with Chagas disease, even minor effects can make a difference in the individual diagnosis.

Comparative Evaluation of Real-Time Screening PCR Assays for Giardia duodenalis and of Assays Discriminating the Assemblages A and B.

Due to superior sensitivity compared to traditional microscopy, real-time PCR has been well established for the diagnosis of Giardia duodenalis in human stool samples. In this study, screening real-time PCRs for different target genes of G. duodenalis, i.e., the 18S rRNA gene, the gdh (glutamate dehydrogenase) gene and the bg (beta-giardin) gene, were comparatively assessed next to various real-time PCR assays for the discrimination of the assemblages A and B of G. duodenalis targeting the bg gene with and without locked nucleic acid-containing probes as well as the tpi (triose phosphate isomerase) gene. The screening PCRs were assessed by including 872 non-preselected samples with a high pre-test probability for G. duodenalis in the statistical analysis, while 53 G. duodenalis-positive samples as indicated by at least two screening PCRs were finally included in the assessment of the assemblage-specific PCRs. For the screening PCRs, sensitivity estimated with latent class analysis (LCA) ranged from 17.5% to 100%, specificity from 92.3% to 100% with an accuracy-adjusted prevalence of 7.2% for G. duodenalis within the non-preselected sample collection. In detail, sensitivity and specificity were 100% and 100% for the 18S rRNA gene-specific assay, 17.5% and 92.3% for the gdh gene-specific assay, and 31.7% and 100% for the bg gene-specific assay, respectively. Agreement kappa was slight with only 15.5%. For the assemblage-specific PCRs, estimated sensitivity ranged from 82.1% to 100%, specificity from 84.0% to 100% with nearly perfect agreement kappa of 90.1% for assemblage A and yet substantial agreement of 74.8% for assemblage B. In detail for assemblage A, sensitivity and specificity were 100% and 100% for the bg gene-specific assay without locked nucleic acids (LNA) as well as 100% and 97.8% for both the bg gene-specific assay with LNA and the tri gene-specific assay, respectively. For assemblage B, sensitivity and specificity were 100% and 100% for the bg gene-specific assay without LNA, 96.4% and 84.0% for the bg gene-specific assay with LNA, and 82.1% and 100% for the tri gene-specific assay, respectively. Within the assessed sample collection, the observed proportion comprised 15.1% G. duodenalis assemblage A, 52.8% G. duodenalis assemblage B and 32.1% non-resolved assemblages. Only little differences were observed regarding the cycle threshold (Ct) values when comparing the assays. In conclusion, best diagnostic accuracy was shown for an 18S rRNA gene-specific screening assay for G. duodenalis and for a differentiation assay discriminating the G. duodenalis assemblages A and B by targeting the bg gene with probes not containing locked nucleic acids. By adding additional highly specific competitor assays for confirmation testing, diagnostic specificity can be further increased on the cost of sensitivity if optimized specificity is desired.

The Clinical Features and Immunological Signature of Cyclospora cayetanensis Co-Infection among People Living with HIV in Ghana.

Background: There is a paucity of information on the contemporary burden, disease patterns, and immunological profile of people living with HIV who are co-infected with C. cayetanensis in the post-antiretroviral therapy era. Methods: For this cross-sectional study, stool samples of 640 HIV-positive and 83 HIV-negative individuals in Ghana were tested for C. cayetanensis. Additionally, sociodemographic parameters, clinical symptoms, medical drug intake, and immunological parameters were assessed. Results: The prevalence of C. cayetanensis was 8.75% (n = 56) in HIV-positive and 1.20% (n = 1) in HIV-negative participants (p = 0.015). Within the group of HIV-positive participants, the prevalence reached 13.6% in patients with CD4+ T cell counts below 200 cells/µl. Frequencies of the clinical manifestations of weight loss and diarrheal disease were significantly higher in patients with C. cayetanensis compared to those without co-infection (36.36% vs. 22.59%, p = 0.034 and 20.00% vs. 4.90%, p < 0.001, respectively). The expression of markers of immune activation and exhaustion of T lymphocyte sub-populations was significantly elevated in patients colonized with C. cayetanensis. Conclusions: In the modern post-combined antiretroviral therapy (cART) era, the acquisition of C. cayetanensis among PLWH in Ghana is driven largely by the immunosuppression profile characterized by high expression of markers of immune activation and immune exhaustion.

Screening for Schistosoma spp. and Leishmania spp. DNA in Serum of Ghanaian Patients with Acquired Immunodeficiency.

Both Schistosoma spp. (species) and Leishmania spp. are prevalent in Ghana in West Africa. However, little is known about their local occurrence in immunocompromised individuals. In the study presented here, the real-time PCR-(polymerase chain reaction-)based screening for repetitive DNA (deoxyribonucleotide acid) sequences from the genomes of Leishmania (L.) spp. and Schistosoma (S.) spp. was performed in the serum of HIV-(human immunodeficiency virus-)infected Ghanaian patients. In 1083 assessed serum samples from HIV-positive and HIV-negative Ghanian patients, Leishmania spp.-specific DNA was not detected, while the diagnostic accuracy-adjusted prevalence estimation suggested a 3.6% prevalence of the S. mansoni complex and a 0.5% prevalence of the S. haematobium complex. Associations of schistosomiasis with younger age, as well as with the male sex, could be shown but not with an HIV status. Weakly significant signals for the associations of schistosomiasis with an increased viral load, reduced CD4+ (CD = cluster of differentiation) T cell count, and a reduced CD4+/CD8+ ratio could be observed but was inconsistently lost in the case of the stratification on the species complex level. So, it is concluded that factors other than HIV status are more likely to have influenced the occurrence of Schistosoma spp. infections in the assessed Ghanaian patients. Potential associations between HIV infection-associated factors, such as the viral load and the immune status of the patients, for which weak signals were observed in this hypothesis-forming retrospective assessment, should be confirmed by prospective, sufficiently powered investigations.

Multicentric Evaluation of SeeGene Allplex Real-Time PCR Assays Targeting 28 Bacterial, Microsporidal and Parasitic Nucleic Acid Sequences in Human Stool Samples.

Prior to the implementation of new diagnostic techniques, a thorough evaluation is mandatory in order to ensure diagnostic reliability. If positive samples are scarcely available, however, such evaluations can be difficult to perform. Here, we evaluated four SeeGene Allplex real-time PCR assays amplifying a total of 28 bacteria, microsporidal and parasitic nucleic acid sequence targets in human stool samples in a multicentric approach. In the assessments with strongly positive samples, sensitivity values ranging between 13% and 100% were recorded for bacteria, between 0% and 100% for protozoa and between 7% and 100% for helminths and microsporidia; for the weakly positive samples, the recorded sensitivity values for bacteria ranged from 0% to 100%; for protozoa, from 0% to 40%; and for helminths and microsporidia, from 0% to 53%. For bacteria, the recorded specificity was in the range between 87% and 100%, while a specificity of 100% was recorded for all assessed PCRs targeting parasites and microsporidia. The intra- and inter-assay variations were generally low. Specifically for some helminth species, the sensitivity could be drastically increased by applying manual nucleic acid extraction instead of the manufacturer-recommended automatic procedure, while such effects were less obvious for the bacteria and protozoa. In summary, the testing with the chosen positive control samples showed varying degrees of discordance between the evaluated Allplex assays and the applied in-house reference assays associated with higher cycle threshold values in the Allplex assays, suggesting that samples with very low pathogen densities might be missed. As the targeted species can occur as harmless colonizers in the gut of individuals in high-endemicity settings as well, future studies should aim at assessing the clinical relevance of the latter hint.

Comparison of Three Real-Time PCR Assays for the Detection of Cyclospora cayetanensis in Stool Samples Targeting the 18S rRNA Gene and the hsp70 Gene.

Diagnostic real-time PCR for the detection of Cyclospora cayetanensis in human stool samples has been applied for two decades. However, recent comparative assessments between in-house and commercial assays suggested room for improvement regarding the agreement of positive signals of the applied real-time PCRs. In order to assess the effect of the choice of the target sequence, 3 inhouse real time PCR assays targeting the 18S rRNA gene (n = 2, one of them later referred to as SSU rRNA gene assay to avoid confusion) and the hsp70 gene of C. cayetanensis were compared in a head-to-head comparison with 905 samples with high pretest probability for C. cayetanensis infections from Ghanaian HIV patients in a test comparison without a reference standard. Only slight agreement kappa of 0.095 was observed. In the assays targeting the SSU rRNA gene, the 18S rRNA gene, and hsp70, positive signals were recorded in 63, 45, and 0 instances, respectively, with latent class analysis-based estimation of sensitivity of 32.2%, 23.3%, 0% as well as of specificity of 99.7%, 99.9% and 100%, respectively. High cycle threshold values with an average of about 35 indicated low quantities of target DNA in the samples with similar Ct values in concordantly and discordantly positive samples. In conclusion, the study suggested target-gene-specific differences in the diagnostic accuracy of real-time PCR-based diagnosis of C. cayetanensis as well as an ongoing need for further standardization of this diagnostic approach.

Limited Reliability of the Molecular Detection of Plasmodium spp. from Incubated Blood Culture Samples for Forensic Purposes.

The suitability of incubated blood culture material for forensic molecular malaria diagnosis was assessed for non-endemic settings for cases in which the differential diagnosis malaria was initially overlooked. For the proof-of-principle assessment, residual blood culture materials from febrile patients from tropical Ghana were investigated by real-time PCR and compared with available historic microscopic results. In 2114 samples, for which microscopical results and real-time PCR results were available, microscopical results comprised 711 P. falciparum detections, 7 P. malariae detections, 1 microscopically not-further-discriminable Plasmodium spp. detection as well as 13 detections of mixed infections comprising 12 cases of P. falciparum/P. malariae co-infections and 1 case of a P. falciparum/P. ovale complex co-infection, while real-PCR indicated 558 P. falciparum detections, 95 P. malariae detections, 10 P. ovale complex detections, 1 P. vivax detection and 4 detected P. falciparum/P. malariae co-infections. Concordance of routine microscopy and real-time PCR was imperfect. Using routine microscopy as reference was associated with a seemingly low agreement of positive real-time PCR results of 90.9%. However, if positive samples, either by routine microscopy or real-time PCR or both, were applied as a combined reference, the agreement of positive results obtained with real-time PCR was increased from 74.0% to 77.9%, while the agreement of positive results obtained with routine microscopy was decreased from 100% to 85.3%. The predictive value of routine microscopy for negative results in the reference was slightly better with 90.9% compared to real-time PCR with 86.9%; the concordance between routine microscopy and real-time PCR was imperfect. In conclusion, even suboptimal sample materials such as incubated blood culture materials can be applied for forensic malaria diagnosis, if more suitable sample materials are not available, but the molecular detection rate of positive results in routine microscopy is much lower than previously reported for non-incubated blood.

Comparison of Three Real-Time PCR Assays Targeting the SSU rRNA Gene, the COWP Gene and the DnaJ-Like Protein Gene for the Diagnosis of Cryptosporidium spp. in Stool Samples.

As qualified microscopy of enteric parasitoses as defined by high diagnostic accuracy is difficult to maintain in non-endemic areas due to scarce opportunities for practicing with positive sample materials, molecular diagnostic options provide less investigator-dependent alternatives. Here, we compared three molecular targets for the real-time PCR-based detection of Cryptosporidium spp. From a population of 1000 individuals comprising both Ghanaian HIV (human immunodeficiency virus) patients and military returnees after deployment in the tropics, stool samples were assessed for Cryptosporidium spp. by real-time PCR targeting the small subunit ribosomal RNA (SSU rRNA) gene, the Cryptosporidium oocyst wall (COWP) gene, and the DnaJ-like protein gene (DnaJ), respectively. In declining order, sensitivity of 100% for the SSU rRNA gene PCR, 90.0% for the COWP PCR and 88.8% for the DnaJ PCR, respectively, as well as specificity of 99.6% for the COWP PCR and 96.9% for both the SSU rRNA gene PCR and the DnaJ PCR, respectively, were recorded. Substantial agreement (kappa value 0.663) between the three assays was observed. Further, an accuracy-adjusted Cryptosporidium spp. prevalence of 6.0% was calculated for the study population. In conclusion, none of the assessed real-time PCR assays were associated with perfect test accuracy. However, a combination of highly sensitive SSU rRNA gene PCR for screening purposes and more specific COWP PCR for confirmatory testing should allow reliable diagnosis of Cryptosporidium spp. in stool samples even in low prevalence settings.