Abstract
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.
Introduction
Human filarial infections predominantly occur in resource-limited tropical settings, while imported infections in travelers are rare events [1, 2]. Mansonellosis, in particular, is no exemption [2–5]. The disease is caused by the nematode species Mansonella perstans, Mansonella ozzardi and Mansonella streptocerca [3], for which humans represent the main definite hosts. While the helminth larvae are predominantly transmitted by Ceratopogonidae (“biting midges”), Simuliidae (“black flies”) have been described to specifically serve as vectors for M. ozzardi [3]. Both vectors are of small size and show outdoor-biting habits [6]. Transfusion-associated transmission occurs but is a very rare event [7]. Human mansonellosis is thought to be asymptomatic or characterized by non-specific symptoms like itching, joint pain, leg chills, enlarged lymphatic nodes, headaches, febrile body temperature and diffuse abdominal discomfort [3, 6, 8]. Symptoms may be associated with the presence of microfilaria in peripheral blood or migration of adult worms [3, 6, 8]. However, neurological manifestations have been reported as well [9] next to corneal lesions caused by M. ozzardi [3]. Therapy of human mansonellosis is insufficiently standardized. Reported therapeutic schemes comprise ivermectin or levamisole-mebendazole-combinations against Mansonella spp. microfilaria, diethylcarbamazine-mebendazol-combination therapy against M. perstans microfilaria, diethylcarbamazine-ivermectin-combination therapy against M. streptocerca microfilaria as well as doxycycline against Wolbachia spp. endosymbionts within some M. perstans and M. ozzardi strains [1, 3]. In Sub-Saharan Africa, M. perstans and M. streptocerca can be found, while in Latin America and the Caribbean, M. ozzardi- and M. streptocerca-associated infections occur [3].
Ghana is one of the Western African countries where filarial infections are still prevalent. As a consequence of control programs, however, onchocerciasis and lymphatic filariasis prevalence in Ghana has dropped to less than 1% [10–12] over the recent decades from initial high percentages [13, 14] in various Ghanaian regions. Nevertheless, those filarial diseases still occur in Ghana with hints for considerable clonal diversity [15], next to Mansonella spp. infections [16–18]. For M. perstans, regional prevalence of about 32% has been reported for Ghanaian individuals with ranges between 2% and 75% and associations with rural, swampy areas providing optimum breeding conditions for transmitting vectors [19]. Accordingly, Mansonella infections are still associated with a relevant infection pressure in Ghana.
Little is known about epidemiological associations of mansonellosis and other infectious diseases in endemicity settings as well as about potential interactions with co-infections. In a recent assessment, co-infections of filarial infections and malaria were only rarely observed [20]. In central African regions of co-endemicity, however, co-infections with Loa loa and Mansonella spp. were recently described to be frequent events [21]. In the study presented here, occurrence of cell-free M. perstans DNA in the peripheral blood serum of predominantly HIV-(human immunodeficiency virus-)positive Ghanaian individuals was assessed to contribute to the scarcely available knowledge on co-infections in mansonellosis patients.
Materials and methods
Study type
The study was conducted as a retrospective cross-sectional assessment.
Study population
To investigate potential associations between infections with the human immunodeficiency virus (HIV) and M. perstans infections, residual serum samples collected in the course of previous studies [22, 23] from 989 HIV positive and 91 HIV negative Ghanaian individuals were assessed. The mean age (± standard deviation (±SD)) within the study population was 39.6 (±9.9), the female:male ratio was 1:3. Within the HIV-positive subpopulation, the median CD4+ T-cell count/µL (interquartile range (IRQ)) was 392.5 (189, 610), the median CD4+/CD8+ T-cell ratio (interquartile range (IRQ)) was 0.4 (0.2, 0.7) and the median viral load in log10 copies/mL (interquartile range (IRQ)) was 4.0 (1.6–5.2).
Molecular assessments
All samples were subjected to nucleic acid extraction applying the EZ1 Virus Mini Kit v2.0 assay (Qiagen, Hilden, Germany) on an automated EZ1 Advanced system (Qiagen). Fluorescence resonance energy transfer (FRET) real-time PCR targeting an ITS-(internal transcribed spacer-)2 sequence of M. perstans as well as phylogenetically related Mansonella sp. Deux was conducted as described recently [24 as well as a submitted manuscript by Veletzky et al.]. The protocol was adapted to a RotorGene Q cycler (Qiagen, Hilden, Germany), leading to minor modifications of the protocol. Details are provided in Table 1.
Details on the applied fluorescence resonance energy transfer (FRET) real-time PCR targeting an ITS-(internal transcribed spacer-)2 sequence of Mansonella perstans as well as phylogenetically related Mansonella sp. Deux on a RotorGene Q cycler. Hyphens in the oligonucleotide sequences have been inserted to increase the readability, not to delineate codon triplets
| PCR target and oligonucleotides | |
| PCR target | M. perstans as well as phylogenetically related Mansonella sp. Deux |
| Target gene | ITS-2 sequence |
| Detection limit | <102 copies/µL |
| Forward primer Mans-FRET-ITS-F | 5′-CCT-AAA-CCG-TCG-ATA-ATG-ATG-A-3′ |
| Reverse primer Mans-FRET-ITS-R | 5′-CAC-CGC-TAA-GAG-TTA-AAA-ATT-TC-3′ |
| Probe Mans-FRET-ITS-S and modifications | 5′-LC640-AAT-ACA-CAC-ATA-CAT-ATA-CTA-ATT-GTA-ATT-ATT-GA-3′ |
| Probe Mans-FRET-ITS-A and modifications | 5′-AAT-AAG-CAT-TTA-TGC-TAA-ATA-TGC-TAC-CAA-CAA-AT-AF488-3′ |
| Positive control plasmid sequence insert | 5′-CAA-ATT-ATC-GCC-TAA-ACC-GTC-GAT-AAT-GAT-GAA-GAT-AAA-GCG-ATA-GCT-TAA-TTA-ATT-TTT-TAT-GAA-AAT-TAA-TTA-AGT-AGA-CTT-AAT-AAG-CAT-TTA-TGC-TAA-ATA-TGC-TAC-CAA-CAA-ATA-AAT-ACA-CAC-ATA-CAT-ATA-CTA-ATT-GTA-ATT-ATT-GAA-AAA-AAC-ATT-AAA-GAA-ATT-TTT-AAC-TCT-TAG-CGG-TGG-ATC-ACT-TGG-3′ |
| GenBank accession number of the insert | MN432520.1 |
| Reaction chemistry | |
| Master Mix | HotStarTaq (Qiagen, Hilden, Germany) |
| Reaction volume | 20 µL |
| Forward primer concentration | 40 pmol μL−1 |
| Reverse primer concentration | 40 pmol μL−1 |
| Probe concentration (each) | 30 pmol μL−1 |
| Final Mg2+ concentration | 3.0 mM |
| Run conditions | |
| Initial denaturation | 15 min at 95 °C |
| Cycle numbers | 45 |
| Denaturation | 20 s at 95 °C |
| Annealing | 45 s at 60 °C (touchdown 5 × 0.5 °C/cycle; fluorescence measurement at this step) |
| Amplification | 20 s at 72 °C |
| Hold | 2 min at 95 °C followed by 30 s at 45 °C |
| Melting conditions | Ramp from 45 °C to 80 °C, 1 °C rising at each step, 90 s pre-melt conditioning at first step and 4 s pre-melt conditioning for each subsequent step |
| Expected melting temperature (Tm) range | 62.3–63.3 °C |
| Cooldown | 30 s at 40 °C |
Plasmid-based positive controls and PCR-grade water-based negative controls were included in each real-time PCR run. Each typically sigmoid-shaped amplification curve, associated with a melting temperature (Tm) within the expected temperature range (Table 1), was considered as indicative of a positive result irrespective of the measured cycle-threshold value. Ten-fold dilution steps of the positive control plasmid were used to calculate the technical detection threshold applying the software “Copy number calculator for real-time PCR” (https://scienceprimer.com/copy-number-calculator-for-realtime-pcr, last accessed on 23rd August 2023). The calculated technical detection threshold was 41 copies/µL. Sample inhibition was controlled applying an inhibition control PCR targeting Phocid Herpes virus DNA [25].
Ethics
The samples were collected and analyzed based on protocols approved by the Committee on Human Research of the Kwame Nkrumah University of Science and Technology in Kumasi, Ghana (reference code CHRPE/AP/12/11; 8th September 2012) and the ethics committee of the Medical Council in Hamburg, Germany (reference code PV3771) in line with the Declaration of Helsinki and all its amendments.
Results
Interestingly, only two positive real-time PCR signals were recorded, with moderate Ct values of 26 and 31 and corresponding melting temperatures of 63.2 and 62.5 °C within the expected melting temperature range.
These two positive real-time PCR signals were observed in two male HIV-positive individuals between 30 and 40 years of age who were without antiretroviral therapy at the time of sample acquisition. None of these two individuals from an urban population worked in agriculture as a potential exposition risk. In one instance, the initial HIV diagnosis was made less than one month prior to the sample acquisition. This patient had a low CD4+ T cell count of less than 100/µL. The other patient, whose initial HIV diagnosis had been established one year prior to the sample acquisition, had still a normal CD4+ cell count but his infection was associated with a high viral load.
In the group of the Ghanaian HIV-negative control individuals, no hint for Mansonella-specific DNA was detected in the serum samples.
Discussion
The study was conducted to assess the prevalence of cell-free M. perstans DNA in serum samples obtained from Ghanaian HIV-patients and revealed several results.
First, compared to described Mansonella prevalence rates in Ghana [19], the prevalence of circulating pathogen DNA in the assessed sera of Ghanaian HIV patients was surprisingly low. The urban setting of the assessed study population might be a potential reason, as Mansonella prevalence rates show a wide geographic variation in Ghana [19]. Although real-time PCR assessments from EDTA (ethylenediaminetetraacetic acid) blood should be generally preferred because of higher sensitivity due to their coverage not only of cell-free but also of cell-bound parasite DNA, the general feasibility of highly sensitive molecular diagnostic approaches for the detection of cell-free filarial DNA in human serum has previously been shown at least for other filarial species like Dirofilaria immitis and Wuchereria brancrofti [26, 27]. As known from other blood-borne helminths like Schistosoma spp. [28, 29], multi-copy PCR-targets with ultra-high copy numbers provide best sensitivity for screenings targeting cell-free helminth DNA in serum. However, multi-copy targets with only moderate copy numbers like the internal transcribed spacer element, which was also used in the here-presented assessment, showed only a minor shift in cycle threshold values in a recent schistosomiasis study compared to Sm1-7, which is a genomic element with ultra-high copy numbers in the genome of Schistosoma mansoni complex [30].
Internationally available data on associations of Mansonella infections and HIV infections are widely non-conclusive. In a study from Gabon, increased Mansonella prevalence had been recorded in HIV patients, particularly in individuals with low CD4+ T cell counts [31], which is in striking contrast to the here-presented results from Ghana. Also, a higher prevalence of Kaposi sarcoma-associated Herpes virus in HIV-positive individuals infected with Mansonella has been reported [32] and a case of a severe Mansonella infection affecting the bone marrow of a HIV patient has been demonstrated [33]. However, those findings suggesting a link between Mansonella infections and immunosuppression in HIV patients are in contrast to a report from Uganda, describing an association of Mansonella infections and even higher CD4+ T cell counts in HIV-infected patients [34]. Obvious interactions between antiretroviral therapy and helminth infections had not been seen in a recent study addressing this particular question [35] and also, HIV-related mycobacterial infections do not seem to be relevantly influenced by co-existing Mansonella infections [36].
Both the reasons for the low prevalence of cell-free M. perstans-DNA observed and a potential association with the HIV infection remain unresolved for the assessed Ghanaian population. Due to the completely lacking proof of Mansonella infections in the few assessed non-HIV-positive individuals, no reliable comparison is feasible.
The study has a few limitations. First, the number of assessed cases was shown to be insufficient for a meaningful comparison of HIV-positive and HIV-negative patients. Second, only real-time PCR from serum targeting freely circulating helminth DNA could be used for this retrospective assessment, which limits comparisons to previous studies applying other diagnostic strategies. As explained in more detail above, PCR from full blood rather than just from serum will necessarily have the advantage of higher sensitivity, because DNA from helminth cells and not just circulating free DNA will be prepared for the molecular assessment during the nucleic acid extraction process from full blood.
Conclusions
In spite of the above-mentioned limitations, the assessment indicated a very low prevalence of circulating M. perstans DNA in the serum of Ghanaian HIV patients. Insofar, the presented data do not support assumptions on hypothetical associations between HIV infections and mansonellosis, at least not regarding worm-loads associated with PCR-detectable cell-free Mansonella DNA amounts. Future studies addressing associations of HIV infections and Mansonella infections in Ghana should be sufficiently powered, focused on local areas of high endemicity and comprise additional diagnostic approaches like microscopy and PCR from full blood.
Author contributions
K.A.E., L.V. and H.F. jointly planned the here-presented study. F.W. and H.F. conducted the assessments. K.A.E., R.O.P., F.S.S., T.F., A.D., S.O.A. and R.B. provided the sample acquisition for the study, J.V. the PCR protocol. H.F. wrote the initial manuscript draft. All authors jointly optimized the manuscript revision and approved the final version.
Conflicts of interest
Nothing to declare. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
Funding sources
The reported PCR assessment for M. perstans did not receive external funding. The study implementation was supported by the ESTHER Alliance for Global Health Partnerships and the German Federal Ministry of Education and Research (Project No. 01KA1102). Parts of the molecular diagnostic approach (nucleic acid extraction) were funded by grant 36K2-S-45 1922 “Evaluation and optimization of molecular diagnostic tests for tropical parasitic diseases for surveillance and risk assessment purposes in tropical deployment settings—a German–French cooperation project between the German Armed Forces Hospital Hamburg and the Military Hospital Laveran, Marseille” of the German Ministry of Defense (MoD) awarded to Hagen Frickmann.
Acknowledgements
Annett Michel and Simone Priesnitz are gratefully acknowledged for excellent technical assistance. All authors are grateful to the nurses and physicians of the HIV clinic and the blood bank of the Komfo Anokye Teaching Hospital in Kumasi, Ghana, for their support.
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