We analyzed 279 anonymous blood samples that had been collected during a trial of intermittent preventive malaria treatment for infants from January 2004 through September 2005 (10). Samples came from infants from 9 villages in the rural Afigya Sekyere district, Ashanti region, Ghana, where estimated prevalences of HIV-1 and HCV in adults were <3% (10) and 2.5% (11), respectively. For storage under tropical conditions, an equal volume of whole blood in EDTA was supplemented with buffer AS1 (QIAGEN, Hilden, Germany). DNA isolation was conducted on a BioRobot M48 workstation (QIAGEN), using the MagAttract M48 DNA Mini Kit (QIAGEN) as recommended (sample input volume 200 µL, elution volume 200 µL).

All samples were first tested by a previously described real-time PCR designed to detect established PARV4 genotypes 1 and 2 (12). When initial sequencing identified novel PARV4 genotype 3, a specific real-time PCR for this genotype was developed by using primers 5′-ACCAAGGACACCAGACAGTCTT-3′ and 5′-ACGTGTTCAGACCAAAAGGAT-3′ and probe 5′-FAM-CCAGCTCCATACCTTTCAGCAGTTGC-BHQ1-3′. All samples were retested by using a plasmid-based standard derived from the real-time PCR amplicon of sample Ghana19 for absolute quantification of genome copy numbers. The lower limit of detection of this assay was 10 plasmid copies/reaction. Procedures to prevent PCR contamination were strictly adhered to, and negative controls were used throughout.

In total, 24 (8.6%) of 279 samples were positive for PARV4 genotype 3 DNA. At the time of blood collection, no infant had signs of acute infection (fever, rash, myalgia). Positive samples were found for infants in 7 of 9 studied villages, indicating widespread prevalence. To test whether socioeconomic factors might influence prevalence, we conducted a χ² test. A significantly lower relative risk of acquiring PARV4 infection was found for children who had access to a kitchen (indicative of higher level of hygiene within household) and who did not live close to a river (Table).

Two age groups were randomly selected from the cohort: 1) 94 infants with a median age of 14.9 months (interquartile range 14.5–15.5 months; 47 male, 47 female) and 2) 185 infants with a median age of 24.0 months (interquartile range 23.5–24.2 months; 92 male, 93 female). Significantly more positive results were seen among infants in the older versus the younger age group (22/185 [11.9%] vs. 2/94 [2.1%], respectively; p<0.006). Viral loads in the whole study group ranged from 420 to 56,000 copies/mL of whole blood (median 3,400 copies/mL). Median viral loads did not differ significantly between the age groups (4,300 vs. 3,200 copies/mL).

A 746-nt fragment of open reading frame (ORF) 1 and a 558-nt fragment of the ORF2 gene and the noncoding region between them were sequenced. Maximal nucleotide distances from previously known PARV4 sequences were 7%–8%. To obtain highly informative sequence datasets for phylogenetic analysis, we concatenated ORF1 and 2 sequences and excluded interfragment recombination by using SimPlot and GARD analysis with the HYPHY package (13). We then conducted phylogenetic analysis on the concatenated fragments by using the neighbor-joining method (Figure). The novel viruses from Ghana clustered in a monophyletic clade with the Nigerian PARV4 genotype 3 prototype strain NG-OR. Within this clade, sequences 1, 3, 4, 10, 12, 13, 17, 18, 19, 20, 21, 23, and 24, sampled from the 2 neighboring villages Asamang and Kona, formed a separate monophyletic subclade, suggesting local epidemic transmission of a unique virus lineage.

Although infection with human parvovirus PARV4 has been considered to be restricted to adults and transmitted parenterally, we found high prevalence of PARV4 genotype 3 in blood of infants in Africa. In agreement with findings for the 2 adults with PARV4 genotype 3 infection (9), clinical signs in these children were not overt. PARV4 infection might thus be clinically silent, or acute infection might be followed by low-level viremia that is cleared slowly, similar to infection with B19.

Finding PARV4 genotype 3 over a 2-year period in several villages suggests common and ubiquitous prevalence. Parenteral medical treatment could be clearly ruled out as a transmission route for the viremic children >2 months of age because these children were under medical observation. It could not be ruled out for younger children; however, because such treatment is uncommon in the rural Ashanti region, it is unlikely. The possibility of virus transmission by vaccination with inadequately sterilized needles is excluded because vaccination was conducted with single-used syringes. Finding virus in persons without parenteral exposure overlaps with a recently raised suspicion regarding a different epidemiology of PARV4 infection in Africa as opposed to the Northern Hemisphere (9).

High prevalence in children in Africa contrasts with the prevalence pattern in the Northern Hemisphere, suggesting different dynamics and routes of transmission. Prenatal or perinatal transient infection can be largely ruled out because the older children had substantially higher rates of viremia. On the contrary, our analysis of socioeconomic factors identified a reciprocal association of transmission risk with access to a kitchen and distance from a river. Foodborne or smear transmission (contact with contaminated objects) are thus suspected. In conclusion, in Africa, novel PARV4 genotype 3 is prevalent among infants who are most likely not at risk for parenteral exposure.