Study sites and participants

During April 2011–November 2012, patients aged ≥5 years admitted with AUF were consecutively included from the following secondary, community (100 to 500) bed hospitals: Baptist Christian Hospital in Tezpur (Assam, North East India), Duncan Hospital in Raxaul (Bihar, North India), Christian Hospital in Mungeli (Chhattisgarh, Central India), B.K. Walawalkar Hospital in Ratnagiri (Maharashtra, Western India), Rural Development Trust Hospital in Anantapur (Andhra Pradesh, South India), Christian Fellowship Hospital in Oddanchatram (Tamil Nadu, South India) and Bethesda Hospital in Ambur (Tamil Nadu, South India) (Fig. 1).

Details of the climate variation among the study sites have been published previously [7]. The study coordinating centre was Christian Medical College (CMC), Vellore, India.

AUF was defined as measured temperature ≥ 38 °C and history of febrile illness of 2–14 days duration, with no localized cause as judged by the treating physician. Patients were not excluded if they had abdominal pain, diarrhoea, haematochezia, nausea or vomiting, rhinorrhoea, dyspnoea, ocular pain, altered sensorium, headache, stiff neck, rash, arthralgia, myalgia, petechiae, ecchymosis, epistaxis, gingival bleeding or jaundice.

Study procedures

Statistical analysis

Chi-square test was used to assess differences between proportions.

Overall results based on case definitions

As per case definition, malaria positivity was found in 17% (268/1564), dengue in 16% (244/1564), scrub typhus in 10% (159/1564), bacteraemia in 8% (124/1564), leptospirosis in 7% (116/1564) and chikungunya in 6% (98/1564). Among malaria cases, 54% (145/268) were Plasmodium falciparum. Details of malaria results in this study have been reported previously [7]. Among bacteraemia cases, Salmonella typhi or S. paratyphi constituted 35% (44/124), Staphylococcus aureus 19% (24/124), E. coli 9% (11/124) and Streptococcus pneumoniae 6% (7/124).

Centre-wise aetiologies

The highest prevalence of malaria was found in West-, North- and Central India (Ratnagiri 34%, Raxaul 28% and Mungeli 21%). The highest prevalence of dengue was found in South- and West India (Anantapur 34%, Ambur 19% and Ratnagiri 30%), and a high prevalence of chikungunya (18%) was also found in Anantapur. The highest prevalence of scrub typhus was found in North- and North East India (Tezpur 22% and Raxaul 15%). Tezpur also had the highest prevalence of leptospirosis (15%). Raxaul in North India had as high prevalence as 20% of bacteraemia.

Overlapping aetiologies

Performance of test systems

Using IFA as gold standard, positive predictive value for Scrub typhus IgM ELISA was 61% (159/260), and negative predictive value was 89% (95/107).

Compared to MAT, positive predictive value for Leptospira IgM ELISA was 65% (116/179) while negative predictive value was as low as 38% (20/52). The study was not designed to evaluate sensitivity and specificity, as gold standard tests were not performed on all ELISA negatives.

Using malaria PCR as gold standard, the sensitivity of routine microscopy was 29% (66/228) and RDT 24% (65/268), as reported previously [7].

Sensitivity and specificity for dengue tests were not calculated because gold standard was positive RDT and/or MAC ELISA, and the two tests are expected to be positive during different intervals of the illness. Indeed, only 46% (57/124) of RDT positives were positive by MAC ELISA, probably reflecting early infections detected only by NS1Ag.

Malaria

Malaria parasites were detected by PCR in 17% (268/1564) among patients included, and among these 54% (145/268) were P. falciparum, as reported previously [7]. Due to high sensitivity of malaria PCR compared to microscopy and RDT, some PCR positive cases may potentially have had asymptomatic low parasitemia controlled by immunity, or recently been treated for malaria, and their fever caused by another infection [7, 9]. As reported previously, microscopy had low sensitivity (29%, 66/228) but high specificity (98%, 918/940) compared to PCR, and a very strict case definition of clinical malaria as cause of acute fever can be defined as a positive microscopy confirmed by PCR [7]. The prevalence of malaria by microscopy confirmed by PCR was 6% (66/1168).

Bacteraemia

Blood stream infection with pathogenic bacteria was diagnosed in 8% (124/1564), and among these Salmonella typhi or S. paratyphi were found in 35% (44/124), reflecting the high prevalence of enteric fever in India. Enteric fever is closely associated with poor sanitation, lack of safe water supply and treatment failures due to antimicrobial resistance and is still reported as the most common blood stream infection in India and in South Asia [10–13]. The second most common microbe identified was S. aureus (19% 24/124), followed by E.-coli (9%, 11/124) and S. pneumoniae (6%, 7/124).

Dengue

Dengue and severe dengue because of immune enhancement due to a previous infection with another serotype is an increasing problem in India [14, 15]. India is estimated to contribute 34% (33/96 million) of the total global burden of dengue [16], with increasing incidence both of dengue and outbreaks of severe dengue [17, 18]. The risk of severe dengue is high, as more than 25% of the population in Delhi has been reported to have had a past infection [17, 19]. In line with the high prevalence reported in previous studies, dengue was found in as much as 16% (244/1564) in the present study, highest in the sites in South- and West India.

Rapid tests combining detection of non-structural protein 1 (NS1) antigen and IgM/IgG are used in routine diagnostics, as they have high sensitivity both during the viremic early phase of infection when NS1 is produced and after more than five days when IgM can be detected [20]. IgM capture ELISA (MAC ELISA) is used as reference method, but is less sensitive than NS1Ag until day five of infection. Case definition used in the present study was therefore a positive test with RDT and/or ELISA, in order not to miss out early infections detected by NS1Ag. However, background positivity is a potential limitation since MAC ELISA can be positive for several months after infection [21]. Although NS1 antigen is less prone to give cross reactivity than IgM antibodies, combination tests have shown some false positive reactions in non-dengue infections, most commonly in chikungunya [20, 21].

Chikungunya

A large outbreak of Chikungunya was reported in Ahmedabad in India in 2006 [22]. Sharing the same vector, chikungunya is likely to occur during dengue outbreaks, and in a study during a dengue outbreak in Delhi in 2010, 10% (66/666) positive chikungunya cases were diagnosed among dengue IgM negative fever patients [23]. Sporadic outbreaks of chikungunya has been reported in India since 1963, in 2006 affecting 13 states with 1.4 million suspected cases [23], with high numbers in Andhra Pradesh, Tamil Nadu and Maharashtra. This supports the finding in the present study of highest prevalence of chikungunya in Anantapur (Andhra Pradesh), Oddanchatram and Ambur (Tamil Nadu) and Ratnagiri (Maharashtra).

Leptospirosis

Leptospirosis is transmitted by urine from infected animals (rats, cattle, pigs) and is endemic particularly in the Andaman and Nicobar group of islands (“Andaman haemorrhagic fever”) [24]. In AUF studies from South- and Northern India, leptospirosis was reported in 3% and 0.1% respectively [11, 12]. In the present study leptospirosis was found in 7% (116/1564), and cases were identified at all study sites.

Culturing Leptospira is unreliable, and the gold standard is therefore serology confirmed by MAT. MAT detects IgM and IgG antibodies against a pool of live antigens from different Leptospira serovars. A MAT titer >100 is considered positive, but a fourfold rise in convalescence titer or a high single acute phase titer (>200–1600 depending on endemicity) supports the diagnosis [25]. Following acute leptospirosis, both IgM ELISA and MAT remain positive for several years after infection, with duration differing between serogroups [26]. In one prospective study from Barbados, positive MAT was found up to 11 years after infection, with highest prevalence after serogroup Autumnalis infection where 20% had MAT titer >800 after four years [26]. In the present study Autumnalis was found in 7%. Leptospira serovar prevalence and distribution in this study has been reported previously [27]. Leptospira IgM ELISA has been reported positive in 40% and 5% one and six years after infection respectively [26]. Discrimination between acute and previous infection in the present study is limited by lack of convalescent samples, and a low MAT cut-off titer of 100. However, high prevalence of antibodies in all study sites suggests that the disease is endemic in the areas.

Scrub typhus

Scrub typhus is transmitted by mites who live on rats. The disease is, similar to leptospirosis, associated with agricultural work and rural dwelling [28]. Two studies have reported prevalence of 14% and 47% among hospitalized febrile patients in North- and South India respectively [11, 12]. The disease is endemic in various parts of India, but underreported [1, 29–35]. In the present study, scrub typhus was found in 10% (159/1564), and the disease was identified at all study sites.

Serology confirmed by IFA, ideally confirmed by rise of titer in convalescent samples and/or by cut-off values based on endemicity, remains the mainstay of diagnostics since isolation of the bacteria is not possible and PCR from blood has low sensitivity [36]. Sensitivity of IFA may be influenced by antigen variation. Usually antigens from three serotypes (Karp, Kato and Gilliam) are used, while additional antigens may be present in different areas [30, 36]. In the present study, discrimination between previous scrub typhus and acute infection is limited by the lack of convalescent samples. Also an optic density (OD) value of 0.5 may be in the lower range and, thereby, in some cases reflect background positivity.

Potential coinfections

Although background positivity or cross reactivity in serology, and potential subclinical infections in malaria, may have given positive test results in some cases, some of the overlapping aetiologies have probably been due to true clinically relevant coinfections.

Coinfections could occur principally by two different mechanisms; by contracting multiple infections at the same time, or increased pathogenicity of a simultaneous subclinical infection due to immune reactions.

The risk of bacterial sepsis is increased in severe malaria, through immune mediated barrier dysfunction in the gut and bacterial translocation, as well as IL-10 mediated decreased control of bacteraemia [37, 38]. In clinical studies, invasive infection, frequently with Salmonella spp. or other Gram-negatives, are found both in P. falciparum and P. vivax malaria [39, 40], which supports the finding of as much as 9% (25/268) bacteraemia among malaria patients in the present study. On the other hand, asymptomatic malaria controlled by immunity may obscure correct diagnosis of bacterial sepsis [41–43], and an undefined proportion of the malaria positive patients among those with bacteraemia may have had subclinical malaria in the present study.

In a study among Thai rice farmers with leptospirosis diagnosed with 4-fold rise in titer or a single high titer, as many as nine among 22 patients had coinfection with scrub typhus confirmed by serology and eschar or clinical characteristics [44]. Although a very high overlap between positive tests for scrub typhus and leptospirosis in the present study suggests background positivity or cross reactivity, a proportion of the patients may have had coinfections taking into consideration the similar exposure risk.

True coinfections with malaria and scrub typhus, diagnosed by clinical characteristics and eschar or PCR, have also been reported in India [45–47]. However, the high level of positive scrub typhus serology in single samples found in other Indian studies [12, 48], raises the same question as in the present study where 10% (27/268) of malaria cases had positive scrub typhus serology, do the results reflect true coinfections, or cross reactivity or background positivity?

In the mosquito borne infections dengue, malaria and chikungunya, outbreaks occur during rainy seasons and although the specific vector is different for malaria, coinfections are not unlikely. This was shown in a study from India during a dengue outbreak, where 7% (27/367) of dengue cases had coinfection with malaria [49]. As much as 22% (58/268) of malaria cases had positive dengue tests in the present study (Tables 3 and 4).

A high level of coinfections with dengue and chikungunya was shown during a dengue outbreak in Delhi in 2006 using PCR as the method for detection. Among 17 chikungunya positive patients, six were co-infected with dengue virus [50]. Ten percent coinfection was found in a study from Mumbai [51]. Dengue and chikungunya virus share a common mosquito vector, the daytime biting Aedes aegypti and A. albopictus, and are present in similar geographical regions. In the present study, dengue and chikungunya both had high prevalence in Anantapur, supporting the notion that coinfections as well as cross reactivity could explain some overlap between dengue and chikungunya.