Outstanding insecurities concerning the use of an Ov16-based ELISA in the Amazonia onchocerciasis focus

Abstract

In a recent issue of Memórias do Instituto Oswaldo Cruz, published in Rio de Janeiro in February 2014 (109: 87-92), Adami et al. have published a survey reporting Mansonella parasite prevalence in the Amazon Region. This report makes a useful contribution to the existing knowledge of filarial parasite distribution within the Amazon area, parasite prevalence rates in relation to age and occupation and provides observations on the possible clinical impact of Mansonella ozzardi. Their publication also provides an account of what appears to be a novel ELISA that has recently been used in the Simuliidae and Onchocerciasis Laboratory of the Oswaldo Cruz Institute, Rio de Janeiro, Brazil. We are concerned that the publication of this ELISA may have created an excessively positive impression of the effectiveness of the onchocerciasis recrudescence serological surveillance tools that are presently available for use in the Amazonia onchocerciasis focus. In this letter we have, thus, sought to highlight some of the limitations of this ELISA and suggest how continuing insecurities concerning the detection of antibodies to Onchocerca volvulus within the Amazonia onchocerciasis focus might be minimised.

Amazonia; ELISA; Ov16; onchocerciasis; Mansonella ozzardi; recrudescence

In a recent issue of Memórias do Instituto Oswaldo Cruz, published in Rio de Janeiro in February 2014 (109: 87-92) [Adami et al. (2014), first available on-line in advance of publication in October 2013] a survey reporting Mansonella parasite prevalence in the Amazon Region has been made. This report makes a useful contribution to the existing knowledge of filarial parasite distribution within the Amazon area, parasite prevalence rates in relation to age and occupation and provides observations on the possible clinical impact of Mansonella ozzardi. Their publication also provides an account of what appears to be a novel ELISA that has recently been used in the Simuliidae and Onchocerciasis Laboratory of the Oswaldo Cruz Institute, Rio de Janeiro, Brazil. We are concerned that the publication of this ELISA could potentially adversely influence the work of the Onchocerciasis Elimination Program for the Americas (OEPA) in the Amazonia onchocerciasis focus. Our concerns about this ELISA are outlined below.

The Amazonia onchocerciasis focus is the last of the Latin American onchocerciasis foci where transmission is still thought to be ongoing and is the last onchocerciasis focus where OEPA expects to eliminate onchocerciasis (Eberhard 2013, WHO/PAHO 2013). Eliminating the disease from this focus presents numerous special challenges including coping with problems arising from the co-existence of M. ozzardi and Onchocerca volvulus parasites within the site, but is nevertheless still expected to occur by 2019 (WHO/PAHO 2013). The detection of antibodies to O. volvulus in children is an important step in OEPA’s published recrudescence surveillance guidelines (Cupp 2012) and has already played a key role in disease monitoring at other Latin American foci (Convit et al. 2013, Rodríguez-Pérez et al. 2013). Presently, OEPA guidelines only recommend Ov16-based serological surveillance be used for this function [Lobos et al. (1991), Lipner et al. (2006) and Lindblade et al. (2007), all in Cupp (2012)], for which M. ozzardi cross-reaction problems are known to exist (Lobos et al. 1991). Although alternative serological monitoring tools could perform the same function, these alternatives also suffer from M. ozzardi cross-reaction problems (Cabrera et al. 1989, Shelley et al. 2001, Post et al. 2003). The novel M. ozzardi-cross-reaction-free O. volvulus ELISA that Adami et al. (2014) have published thus seems, ostensibly, like a timely development for the OEPA committee and to fit well with OEPA-published guidelines on recrudescence monitoring (Cupp 2012). We, however, feel that the description of this ELISA is potentially misleading and that outstanding limitations of the assay were not made sufficiently clear. We have, therefore, decided to raise some of our concerns about the Adami et al. (2014) paper here. Our hope is that in doing this World Health Organization time and resources may avoid being misspent.

Our most serious concern with the publication of this (Ov10, Ov11, Ov16) ELISA is that no sensitivity data have been presented with it. In the methodology section of Adami et al. (2014) the authors state that each ELISA plate contained “a reference positive (n = 3, strongly reacting plasma from onchocerciasis patients)”. Whether the authors deemed that their data set of sera positive for antibodies to O. volvulus is insufficiently large or insufficiently representative of what is found in the field as to have any value for sensitivity calculations is unclear. What is clear, however, is that they have published no sensitivity data (with or without predictive values) and that because of this the rest of the ELISA data are of only very questionable value. Certainly, with the information provided by Adami et al. (2014), the ELISA’s potential value cannot be properly measured against the ELISAs described in Lobos et al. (1991) and Bradley et al. (1993) or other important serological surveillance tools like the Lipner et al. (2006) assay which has been used for recrudescence monitoring in other Latin American onchocerciasis foci.

Our second concern with the Adami et al. (2014) paper is that its findings appear to be in direct conflict with findings that Adami et al. (2008) paper. The two papers both report O. volvulus ELISA cross-reactivity data from ELISAs that seem to be based on the same O. volvulus protein cocktail containing the Ov10, Ov11 and Ov16 protein antigens. The results reported from the two studies are, however, very different. In the Adami et al. (2008) paper an O. volvulus ELISA is described as having cross-reacted with 40% of the tested M. ozzardi positive sera from Vila Antimary, whereas the O. volvulus ELISA reported in the Adami et al. (2014) is said to have cross-reacted with none of the M. ozzardi positive sera samples taken from the very same site. While the authors offer an explanation that their change of the Ov29 protein for an Ov16 protein might be the reason why they have not observed the cross reactivity problems that were reported in Shelley et al. (2001), they make no explicit reference to why the same ELISA cocktail they used in 2014 produced such radically different results in 2008 (Adami et al. 2014). If the authors have developed a new protocol to resolve the problems they first encountered with the Ov10, Ov11 and Ov16 protein cocktail that they reported in 2008, this should have been made clearer in their 2014 paper. Similarly, if the Adami et al. (2008) report of ELISA cross-reactivity was erroneous, this should have been made clear in the Adami et al. (2014) paper.

Our final concern with the Adami et al. (2014) paper that we would like to highlight relates to its filarial parasite identifications. Adami et al. (2008) reported the existence of typical and atypical M. ozzardi parasites in the same area in which Adami et al. (2014) have tested their novel ELISA and, indeed, in the Adami et al. (2014) paper it is recorded that the novel ELISA was tested on both types of parasite. While similar reports of morphologically atypical M. ozzardi in Peru have previously been shown to be molecularly identical to typical forms (Marcos et al. 2012), the parasites at the Adami et al. (2014) study site have not yet been shown to be equally homogenous. Given that the authors present a picture of a diverse, transient, fluctuating population of M. ozzardi in their study area, it is disappointing that they chose not to clarify that the parasites that are presently in their study area are indeed the same parasites that Shelley et al. (2001) reported causing ELISA cross-reactivity problems. There are many existent polymerase chain reaction (PCR)-based filarial parasite identification techniques that they could have easily been adapted to assist with this - see, for example, Morales-Hojas et al. (2001), Post et al. (2009) or Tang et al. (2010).

In light of the outstanding issues relating to O. volvulus ELISA cross-reactivity with M. ozzardi, we recommend that before a serological tool is chosen for recrudescence monitoring in the Amazonia onchocerciasis focus, its specificity and sensitivity should be established and compared against all existing alternatives on samples obtained from within the focus. We recommend that the data generated for each of these tools should be compared directly, with microscopy and PCR evaluations of skin biopsies and blood samples taken from the same individuals. We additionally recommend that if OEPA has not already modified their recrudescence surveillance guidance (Cupp 2012) to take account of potential M. ozzardi ELISA cross-reactivity issues inside the Amazonia onchocerciasis focus, they should consider modifying their serological survey to include a PCR assay of ELISA positive blood samples found at the site. We recommend that a PCR [like that described in Tang et al. (2010)] should be performed on all O. volvulus positive blood samples before skin biopsies are performed, at least until such times as ELISA M. ozzardi cross-reactivity can be properly resolved. Such a modification could minimise the impact of false positives on disease control and planning strategies and could also avoid the trauma caused by unnecessary skin biopsies. We also believe that such a modification could help clarify which tested people may have actually been “exposed” to onchocerciasis.

REFERENCES

• Adami YL, Moraes MAP, Lanfredi RM, Maia-Herzog M 2008. An atypical microfilaria in blood samples from inhabitants of Brazilian Amazon. Parasitol Res 104: 95-99.
• Adami YL, Rodrigues G, Alves MC, Moraes MAP, Banic DM, Maia-Herzog M 2014. New records of Mansonella ozzardi: a parasite that is spreading from the state of Amazonas to previously uninfected areas of the state of Acre in the Purus River region. Mem Inst Oswaldo Cruz 109: 87-92.
• Bradley JE, Trenholme KR, Gillespie AJ, Guderian R, Titanji V, Hong Y, McReynolds L 1993. A sensitive serodiagnostic test for onchocerciasis using a cocktail of recombinant antigens. Am J Trop Med Hyg 48: 198-204.
• Cabrera Z, Parkhouse RM, Forsyth K, Gomez Priego A, Pabon R, Yarzabal L 1989. Specific detection of human antibodies to Onchocerca volvulus Ann Trop Med Parasitol 40: 454-459.
• Convit J, Schuler H, Borges R, Olivero V, Domínguez-Vázquez A, Frontado H, Grillet ME 2013. Interruption of Onchocerca volvulus transmission in northern Venezuela. Parasites Vectors 6: 289.
• Cupp EW 2012. Guide to detecting a potential recrudescence of onchocerciasis during the post treatment surveillance period: the American paradigm. Res Rep Trop Med 3: 21-33.
• Eberhard M 2013. Progress toward elimination of onchocerciasis in the Americas - 1993-2012. Morbidity and Mortality Weekly Report. Available from: cdc.gov/mmwr/pdf/wk/mm6220.pdf.
  » cdc.gov/mmwr/pdf/wk/mm6220.pdf
• Lindblade KA, Arana B, Zea-Flores G, Rizzo N, Porter CH, Dominguez A, Cruz-Ortiz N, Unnasch TR, Punkosdy GA, Richards J, Sauerbrey M, Castro J, Catú E, Oliva O, Richards Jr FO 2007. Elimination of Onchocercia volvulus transmission in the Santa Rosa focus of Guatemala. Am J Trop Med Hyg 77: 334-341.
• Lipner EM, Dembele N, Souleymane S, Alley WS, Prevots DR, Toe L, Boatin B, Weil GJ, Nutman TB 2006. Field applicability of a rapid-format anti-Ov-16 antibody test for the assessment of onchocerciasis control measures in regions of endemicity. J Infect Dis 194: 216-221.
• Lobos E, Weiss N, Karam M, Taylor HR, Ottesen EA, Nutman TB 1991. An immunogenic Onchocerca volvulus antigen: a specific and early marker of infection. Science 251: 1603-1605.
• Marcos LA, Arrospide N, Recuenco S, Cabezas C, Weil GJ, Fischer PU 2012. Genetic characterization of atypical Mansonella (Mansonella) ozzardi microfilariae in human blood samples from north-eastern Peru. Am J Trop Med Hyg 87: 491-494.
• Morales-Hojas R, Post RJ, Shelley AJ, Maia-Herzog M, Coscaron S, Cheke RA 2001. Characterisation of nuclear ribosomal DNA sequences from Onchocerca volvulus and Mansonella ozzardi (Nematoda: Filarioidea) and development of a PCR-based method for their detection in skin biopsies. Int J Parasitol 31: 169-177.
• Post RJ, Adams Z, Shelley AJ, Maia-Herzog M, Luna Dias AP, Coscarón S 2003. The morphological discrimination of microfilariae of Onchocerca volvulus from Mansonella ozzardi Parasitology 127: 21-27.
• Post RJ, Crainey JL, Bivand A, Renz A 2009. Laser-assisted microdissection for the study of the ecology of parasites in their hosts. Mol Ecol Resou 9: 480-486.
• Rodríguez-Pérez MA, Domínguez-Vázquez A, Unnasch TR, Hassan HK, Arredondo-Jiménez JI, Orozco-Algarra ME, Rodríguez-Morales KB, Rodríguez-Luna IC, Prado-Velasco FG 2013. Interruption of transmission of Onchocerca volvulus in the southern Chiapas focus, México. PLoS Negl Trop Dis 7: e2133.
• Shelley AJ, Maia-Herzog M, Calvão-Brito R 2001. The specificity of an ELISA for detection of Onchocerca volvulus in Brazil in an area endemic for Mansonella ozzardi Trans R Soc Trop Med Hyg 95: 171-173.
• Tang T-HT, López-Vélez R, Lanza M, Shelley AJ, Rubio JM, Luz SLB 2010. Nested PCR to detect and distinguish the sympatric filarial species Onchocerca volvulus, Mansonella ozzardi and Mansonella perstans in the Amazon Region. Mem Inst Oswaldo Cruz 105: 823-828.
• WHO/PAHO - World Health Organization/Pan American Health Organization 2013. Towards the elimination. In 152nd Session of the Executive Committee, WHO/PAHO, Washington, p. 33-40.

• Financial support: FIOCRUZ, FAPEAM, CNPq
• *
  All papers undertaken by the LSO resulting from either research, collections and reference services are done with total independence and without conflict of interest with relation to their partners or supporters (Brazilian Health Ministry, OEPA) among others.

Publication Dates

• Publication in this collection
  July 2014

History

• Received
  2 Mar 2014
• Accepted
  16 June 2014